Method for using lipoprotein associated coagulation inhibitor to treat sepsis

ABSTRACT

A method for prophylactically or therapeutically treating sepsis or septic shock is described, wherein an inhibitor to tissue factor is administered to septic patients. Additionally, a method for treating inflammation is described wherein the inhibitor is administered to patients. This inhibitor is termed lipoprotein associated coagulation inhibitor, or commonly LACI. It is 38 kD and has 276 amino acids. LACI has now been shown to be useful for the treatment of sepsis, septic shock and inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/368,000, filedFeb. 19, 2003 (abandoned), which is a continuation of U.S. Ser. No.09/971,362, filed Oct. 5, 2001 (abandoned), which is a continuation ofU.S. Ser. No. 09/521,180, filed Mar. 8, 2000 (abandoned), which is acontinuation of Ser. No. 08/472,761, filed Jun. 7, 1995 (now U.S. Pat.No. 6,063,764), which is a continuation-in-part of U.S. Ser. No.08/224,118, filed Mar. 29, 1994 (abandoned), which is a continuation ofSer. No. 08/020,472 filed Feb. 22, 1993 now U.S. Pat. No. 5,368,148,which is a continuation-in-part of Ser. No. 07/897,135, filed Jun. 11,1992 (abandoned). U.S. Ser. No. 08/472,761, filed Jun. 7, 1995 is also acontinuation-in-part of Ser. No. 08/253,427, filed Jun. 2, 1994(abandoned), which is a continuation of Ser. No. 08/004,505 filed Jan.13, 1993 (abandoned), which is a continuation-in-part of Ser. No.07/891,947, filed Jun. 1, 1992 (abandoned). U.S. Ser. No. 08/472,761,filed Jun. 7, 1995 is also a continuation-in-part of Ser. No. 08/270,455filed Jul. 5, 1994 (abandoned), which is a continuation of Ser. No.07/891,947, filed Jun. 1, 1992 (abandoned).

FIELD OF THE INVENTION

The present invention is a method for prophylactically andtherapeutically treating acute and chronic inflammation, sepsis andseptic shock. More specifically, it comprises administering atherapeutically effective amount of a specific protein to attenuatephysiological pathways associated with septic shock.

BACKGROUND OF THE INVENTION

Lipoprotein-associated coagulation inhibitor (LACI) is a proteininhibitor present in mammalian blood plasma. LACI is also known astissue factor (TF) inhibitor, tissue thromboplastin (Factor III)inhibitor, extrinsic pathway inhibitor (EPI) and tissue factor pathwayinhibitor (TFPI).

Blood coagulation is the conversion of fluid blood to a solid gel orclot. The main event is the conversion of soluble fibrinogen toinsoluble strands of fibrin, although fibrin itself forms only 0.15% ofthe total blood clot. This conversion is the last step in a complexenzyme cascade. The components (factors) are present as zymogens,inactive precursors of proteolytic enzymes, which are converted intoactive enzymes by proteolytic cleavage at specific sites. Activation ofa small amount of one factor catalyses the formation of larger amountsof the next, and so on, giving an amplification which results in anextremely rapid formation of fibrin.

The coagulation cascade which occurs in mammalian blood is divided by invitro methods into an intrinsic system (all factors present in theblood) and an extrinsic system which depends on the addition ofthromboplastin. The intrinsic pathway commences when the first zymogen,factor XII or ‘Hageman Factor’, adheres to a negatively charged surfaceand in the presence of high molecular weight kininogen andprekallikrein, becomes an active enzyme, designated XIIa. The activatingsurface may be collagen which is exposed by tissue injury. Factor XIIaactivates factor XI to give XIa, factor XIa activates factor IX to IXaand this, in the presence of calcium ions, a negatively chargedphospholipid surface and factor VIIIa, activates factor X. Thenegatively charged phospholipid surface is provided by platelets and invivo this serves to localize the process of coagulation to sites ofplatelet deposition. Factor Xa, in the presence of calcium ions, aplatelet-derived negatively charged phospholinid surface and a bindingprotein, factor V, activates prothrombin to give thrombin (IIa)—the mainenzyme of the cascade. Thrombin, acting on gly-arg bonds, removes smallfibrinopeptides from the N-terminal regions of the large dimericfibrinogen molecules, enabling them to polymerize to form strands offibrin. Thrombin also activates the fibrin stabilizing factor, factorXIII, to give XIIIa, a fibrinoligase, which, in the presence of calciumions strengthens the fibrin-to-fibrin links with intermolecularγ-glutamyl-ε-lysine bridges. In addition, thrombin acts directly onplatelets to cause aggregation, and release of subcellular constituentsand arachidonic acid. A further function of thrombin is to activate thecoagulation inhibitor, protein C. Factors XIIa, XIa, IXa, Xa, andthrombin are all serine proteases.

The extrinsic pathway in vivo is initiated by a substance generated by,or exposed by, tissue damage and termed ‘tissue factor’, interactingwith Factor VII in the presence of calcium ions and phospholipid toactivate factors X and IX, after which the sequence proceeds as alreadydescribed. The identity of TF is known. There is evidence that tissuefactor occurs in the plasma membranes of perturbed endothelial cells ofblood vessels and also in atheromatous plaques.

The two pathways described are not entirely separate because both factorIXa and factor XIIa in the intrinsic pathway may activate factor VII inthe extrinsic pathway. There are, in addition, various feedback loopsbetween other factors, which enhance reaction rates. For example,thrombin (IIa) enhances the activation of both factor V and factor VIII.

Sepsis and its sequela septic shock remain among the most dreadedcomplications after surgery and in critically ill patients. The Centerfor Disease Control ranks septicemia as the 13th leading cause of deathin the United States (see MMWR, 1987, 39:31 and US Dept. of Health andHuman Services, 37:7, 1989), and the 10th leading cause of death amongelderly Americans (see MMWR, 1987, 39:777). The incidence of thesedisorders is increasing, and mortality remains high. Estimates of thetotal cost of caring for patients with septicemia range from $5 billionto $10 billion annually (see MMWR, 1987, 39:31). Death can occur in 40%to 60% of the patients. This percentage has not seen any improvementover the past 20 years. The incidence of blood borne gram-positive andgram-negative infections that can lead to septic shock occurapproximately equally.

Sepsis is a toxic condition resulting from the spread of bacteria, ortheir products (collectively referred to herein as bacterial endotoxins)from a focus of infection. Septicemia is a form of sepsis, and moreparticularly is a toxic condition resulting from invasion of the bloodstream by bacterial endotoxins from a focus of infection. Sepsis cancause shock in many ways, some related to the primary focus of infectionand some related to the systemic effects of the bacterial endotoxins.For example, in septaemia, bacterial endotoxins, along with othercell-derived materials, such as IL-1, IL-6 and TNF, activate thecoagulation system and initiate platelet aggregation. The process leadsto blood clotting, a drop in blood pressure and finally kidney, heartand lung failure.

Septic shock is characterized by inadequate tissue perfusion, leading toinsufficient oxygen supply to tissues, hypotension and oliguria. Septicshock occurs because bacterial products, principally LPS, react withcell membranes and components of the coagulation, complement,fibrinolytic, bradykinin and immune systems to activate coagulation,injure cells and alter blood flow, especially in the microvasculature.Microorganisms frequently activate the classic complement pathway, andendotoxin activates the alternate pathway. Complement activation,leukotriene generation and the direct effects of endotoxin onneutrophils lead to accumulation of these inflammatory cells in thelungs, release of the enzymes and production of toxic oxygen radicalswhich damage the pulmonary endothelium and initiate the acuterespiratory distress syndrome (ARDS). ARDS is a major cause of death inpatients with septic shock and is characterized by pulmonary congestion,granulocyte aggregation, hemorrhage and capillary thrombi.

Activation of the coagulation cascade by bacterial endotoxins introduceddirectly into the bloodstream can result in extensive fibrin depositionon arterial surfaces with depletion of fibrinogen, prothrombin, factorsV and VIII, and platelets. In addition, the fibrinolytic system isstimulated, resulting in further formation of fibrin degradationproducts. Disseminated intravascular coagulation (DIC) is a complexcoagulation disorder resulting from widespread activation of theclotting mechanism or coagulation cascade which, in turn, results fromsepticemia. Essentially, the process represents conversion of plasma toserum within the circulation system. Such process represents one of themost serious acquired coagulation disorders. Some common complicationsof disseminated intravascular coagulation are severe clinical bleeding,thrombosis, tissue ischaemia and necrosis, hemolysis and organ failure.

At the same time, as coagulation is apparently initiated by endotoxin,countervening mechanisms also appear to be activated by clotting, namelyactivation of the fibrinolytic system. Activated Factor XII convertsplasminogen pro-activator to plasminogen activator which subsequentlyconverts plasminogen to plasmin thereby mediating clot lysis. Theactivation of plasma fibrinolytic systems may therefore also contributeto bleeding tendencies.

Endotoxemia is associated with an increase in the circulating levels oftissue plasminogen activator inhibitor (PAI). This inhibitor rapidlyinactivates tissue plasminogen activator (TPA), thereby hindering itsability to promote fibrinolysis through activation of plasminogen toplasmin. Impairment of fibrinolysis may cause fibrin deposition in bloodvessels, thus contributing to the disseminated intravascular coagulationassociated with septic shock.

Disseminated intravascular coagulation (DIC) is a coagulopathic disorderthat occurs in response to invading microorganisms characterized bywidespread deposition of fibrin in small vessels. The initiating causeof DIC appears to be the release of thromboplastin (tissue factor) intothe circulation. During this process, there is a reduction in fibrinogenand platelets, and a rise in fibrin split products resulting in fibrindeposition in blood vessels. The sequence of events that occur duringDIC are described in FIG. 1. The patients either suffer from thrombosisor hemorrhage depending on the extent of exhaustion of the coagulationprotease inhibitors during the disease process. Part of the regulationof the coagulation cascade depends on the rate of blood flow. When flowis decreased, as it is in DIC and sepsis, the problems are magnified.DIC (clinically mild to severe form) is thought to occur with highfrequency in septic shock patients and several other syndromes such ashead trauma and burns, obstetric complications, transfusion reactions,and cancer. A recent abstract by Xoma Corporation indicates that DIC waspresent on entry in 24% of septic patients (Martin et al., 1989, NaturalHistory in the 1980s, Abstract No. 317, ICAAC Meeting, Dallas).Furthermore, the abstract describes that DIC and acute respiratorydistress syndrome were the variables most predictive of death by day 7(risk ratios 4 and 2.3). The cascade of events that lead to release oftissue factor into circulation and sepsis is very complex. Variouscytokines are released from activated monocytes, endothelial cells andothers; these cytokines include tumor necrosis factor (TNF), interleukin1 (IL-1) (which are known to up-regulate tissue factor expression),interleukin 6 (IL-6), gamma interferon (IFN-γ), interleukin 8 (IL-8),and others. The complement cascade is also activated as demonstrated bythe rise in C3a and C5a levels in plasma of septic patients.Consequently, an agent that will treat coagulation without affecting theexpression of tissue factor or its activity will not necessarily beeffective to treat sepsis.

There are currently no satisfactory interventions for the prevention ortreatment of sepsis or DIC. Heparin is the most commonly usedanticoagulant in DIC. However, it has been controversial because it caninduce bleeding and worsen the patient's condition. See, for example,Corrigan et al., “Heparin Therapy in Septacemia with DisseminatedIntravascular Coagulation. Effect on Mortality and on Correction ofHemostatic Defects”, N. Engl. J. Med., 283:778-782 (1970); lasch et al.,Heparin Therapy of Diffuse Intravascular Coagulation (DIC)”, Thrombos.Diathes. Haemorrh., 33:105 (1974); Straub, “A Case Against HeparinTherapy of Intravascular Coagulation”, Thrombos. Diathes. Haemorrh.,33:107 (1974).

Other attempts to treat sepsis using an anticoagulant have also beendifficult. As shown in Taylor et al., 1991, Blood, 78:364-368, warfarinand heparin are mentioned as two anticoagulants that are used to treatDIC in sepsis, but neither are the ideal drugs. Additionally, Taylor etal. show that a new drug DEGR-Xa, a factor Xa antagonist, can inhibitDIC, however, this drug failed to block the lethal effects of sepsis.Consequently, it is evident that an agent which may interrupt thecoagulation pathway is not necessarily effective as an inhibitor ofseptic shock. Therefore, there is a need in the art for a compositionthat will inhibit the lethal effects of sepsis.

SUMMARY OF THE INVENTION

The present invention is a method for prophylactically andtherapeutically treating syndromes associated with acute or chronicinflammation where activation of Factor VII, Xa and tissue factorexpression are involved, such as sepsis and septic shock, whetheraccompanied by DIC or not. The method comprises administering aneffective amount of lipoprotein associated coagulation inhibitor (LACI).Additionally, the present invention is a method, comprisingadministering LACI, to treat a disease state in which TNF, IL-1, IL-6 orother cytokines up-regulate tissue factor. Specifically, these diseasestates include acute or chronic inflammation. Preferably, LACI isintravenously administered at a dose between 1 μg/kg and 20 mg/kg, morepreferably between 20 μg/kg and 10 mg/kg, most preferably between 1 and7 mg/kg. LACI is preferably administered with an additional agent totreat sepsis and septic shock, such as an antibiotic.

Among other things, it has been surprisingly discovered that a compoundknown for its anti-coagulant properties, can also attenuate the immuneresponse and serve as a treatment for sepsis and septic shock. This wassurprising in view of the findings of Warr et al., 1990, Blood,75:1481-1489 and Taylor et al., 1991, Blood, 78:364-368.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLE

FIG. 1 shows the complex pathways involved in Sepsis and septic shock.The intrinsic and extrinsic pathways are included. Signs ofmicrovascular thrombosis include: (1) neurologic: multifocal, delerium,coma; (2) skin: focal ischemia, superficial gangrene; (3) renal:oliguria, azotemia, cortical necrosis; (4) pulmonary; acute respiratorydistress syndrome; and (5) gastrointestinal; acute ulceration. Signs ofhemorrhagic diathesis include: (1) neurologic: intracerebral bleeding;(2) skin: petechiae, ecchymoses, venepuncture oozing; (3) renal;hematuria; (4) mucous membranes: epistaxis, gingival oozing; and (5)gastrointestinal: massive bleeding.

FIG. 2 shows the inhibition of tissue factor activity by 36 dayconditioned medium (CM) and TNF induced CM.

FIG. 3 shows LACI neutralization of CM from endothelial cells.

FIG. 4 shows antibody neutralization of LACI protein.

FIGS. 5 a and 5 b show pharmacokinetic profile of LACI in baboons. Opencircles represent results in the immunoassay and closed circlesrepresent results in the bioassay. For example, 0.5 mg/kg of LACI wasgiven as an I.V. bolus over 30 seconds to two healthy baboons. Blood wassampled from animals at +1 minute, 3, 6, 10, 20, 40, 60, 90, 120, 180,240 and 420 minutes. LACI levels in plasma were measured using bothimmunoassay and bioassay (described in text). In FIG. 5 b, the linerepresents 0.7 ug/kg+10 ug/kg/min inf. 12 hr.

FIGS. 6 a through 6 h show the coagulation and hematological response toLACI administration 30 minutes after the start of a two hour lethalbacterial intravenous infusion. Lines with solid circles representresults obtained from treated animals and lines with “X”s representresults obtained form control animals. A ★ (star) indicates astatistically significant difference (p<0.05) between the control andexperimental groups and an open circle represents a statisticallysignificant (p<0.05) difference between times. FIG. 6 a shows fibrinogenlevels, FIG. 6 b shows FDP levels, FIG. 6 c shows platelet levels, FIG.6 d shows WBC levels, FIG. 6 e shows PT levels, Figure ^f shows APTTlevels, FIG. 6 g shows hemtocrit levels, and FIG. 6 h shows RBC levels.For example, anesthetized baboons were challenged with a lethal dose ofE. coli (˜5×10¹⁰ organisms/kg) intravenously infused over two hours.Thirty minutes after the start of the bacterial infusion five baboonsreceived phosphate buffered saline (PBS; excipient control; *) and theother five received LACI in PBS (

). Blood samples were obtained from the ten baboons before the start ofthe bacterial infusion, and at 2, 4, 6, and 12 hours after the onset ofinfusion. Blood samples were assayed for fibrinogen, fibrin degradationproducts, prothrombin time, activated partial thromboplastin time, andfor hematocrit, platelet, red cell and white cell counts by standardmethods. Mean ± standard error of each measurement is plotted againsttime (hrs.).

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that LACI in the absence of otheranticoagulants such as heparin is effective in the prophylaxis andtreatment of sepsis. It has also been discovered that LACI alone iseffective in the prophylaxis and treatment of sepsis-associatedcoagulation disorders such as, for example, DIC. LACIinhibits/attenuates the coagulopathies and the inflammatory processassociated with acute inflammatory and septic shock.

LACI is a serum glycoprotein with a molecular weight of 38,000 Kd. It isalso known as tissue factor inhibitor because it is a natural inhibitorof thromboplastin (tissue factor) induced coagulation. (U.S. Pat. Nos.5,110,730 and 5,106,833 describe tissue factor and are herebyincorporated by reference in their entireties). LACI is a proteaseinhibitor and has 3 Kunitz domains, two of which are known to interactwith factors VII and Xa respectively, while the function of the thirddomain is unknown. Many of the structural features of LACI can bededuced because of its homology with other well-studied proteases. LACIis not an enzyme, so it probably inhibits its protease target in astoichiometric manner; namely, one of the domains of LACI inhibits oneprotease molecule. As utilized herein LACI means one or more of the theeKunitz-type inhibitory domains of lipoprotein-associated coagulationinhibitor which are active in treating sepsis. The domains may bepresent on fragments of LACI or in hybrid molecules. See U.S. Pat. No.5,106,833 regarding fragments and muteins. Preferably, Kunitz domains 1and/or 2 will be present. Kunitz domain 3 is not necessary for activity.

LACI is also known as tissue factor pathway inhibitor (TFPI). This namehas been accepted by the International Society on Thrombosis andHemostasis, Jun. 30, 1991, Amsterdam. TFPI was first purified from ahuman hepatoma cell, Hep G2, as described by Broze and Miletich; Proc.Natl. Acad. Sci. USA 84:1886-1890 (1987), and subsequently from humanplasma as reported by Novotny et al., J. Biol. Chem. 264:18832-18837(1989); Chang liver and SK hepatoma cells as disclosed by Wun et al., J.Biol. Chem. 265:16096-16101 (1990). TFPI cDNA molecules have beenisolated from placental and endothelial cDNA libraries as described byWun et al., J. Biol. Chem. 263:6001-6004 (1988); Girard et al., Thromb.Res. 55, 37-50 (1989). The primary amino acid sequence of TFPI, deducedfrom the cDNA sequence, shows that TFPI contains a highly negativelycharged amino-terminus, three tandem Kunitz-type inhibitory domains, anda highly positively charged carboxyl terminus. The first Kunitz-domainof TFPI (amino acids 19 to 89 of mature TFPI and amino acids 47 to 117of pre-TFPI) is needed for the inhibition of the factor VII_(a)/tissuefactor complex and the second Kunitz-domain of TFPI (amino aicds 90 to160 of the mature protein or amino acids 118 to 188 of pre-TFPI) isresponsible for the inhibition of factor X_(a) according to Girard etal., Nature 328:518-520 (1989), while the function of the thirdKunitz-domain (amino acids 182 to 252 of mature TFPI and amino acids 210to 280 of pre-TFPI) remains unknown. See also U.S. Pat. No. 5,106,833.TFPI is believed to function in vivo to limit the initiation ofcoagulation by forming an inert, quaternary factor X_(a): TFPI: factorVII_(a): tissue factor complex. See reviews by Rapaport, Blood73:359-365 (1989), and Broze et al., Biochemistry 29:7539-7546 (1990).

Three truncated versions of LACI have been produced from E. coli. Theseare ala-TFPI-1-160; ala-TFPI-13-161, and ala-TFPI-22-150. Thesederivatives have production advantages and favorable solubilitycharacteristics compared to full-length ala-TFPI (ala-LACI). Thederivatives are produced at levels approximately 7-10 fold higher thanfull-length ala-LACI. Solubility of the derivatives in a physiologicalbuffer, e.g., phosphate buffered saline, is about 40 to 80-fold higherthan full-length ala-LACI. In addition, the clearance rate appearsslower for at least one of the derivatives relative to the full-lengthform. All three forms are active in factor Xa-dependent inhibition offactor VIIa/tissure factor activity. Ala-TFPI-1-160 was tested in ababoon model of sepsis and was found to promote survival. Five of eightanimals treated with the fragment survived to the 7 day endpoint, whilenone of five untreated control baboons survived.

Recombinant TFPI has been expressed as a glycosylated protein usingmammalian cell hosts including mouse C127 cells as disclosed by Day etal., Blood 76:1538-1545 (1990), baby hamster kidney cells as reported byPedersen et al., J. Biol. Chem. 265:16786-16793 (1990), Chinese hamsterovary cells and human SK hepatoma cells. The C127 TFPI has been used inanimal studies and shown to be effective in the inhibition of tissuefactor-induced intravascular coagulation in rabbits according to Day etal., supra, and in the prevention of arterial reocclusion afterthrombolysis in dogs as described by Haskel et al., Circulation84:821-827 (1991).

Recombinant TFPI also has been expressed as a non-glycosylated proteinusing E. coli host cells yielding a highly active TFPI by in vitrofolding of the protein as described below in Example 1.

The cloning of the TFPI cDNA which encodes the 276 amino acid residueprotein of TFPI is further described in Wun et al., U.S. Pat. No.4,966,852, the disclosure of which is incorporated by reference herein.

LACI was discovered by Broze et al., 1987, PNAS (USA), 84:1886-1890, andwas found to inhibit Factor Xa directly, as well as to inhibit tissuefactor activity by formation of an inert factor VIIa/tissue factor(TF)/Factor Xa/Ca++ inhibitor complex. It has the DNA sequence shown inU.S. Pat. No. 4,966,852 which is hereby incorporated by reference in itsentirety. A schematic diagram of the proposed mechanism for theinhibition of Factor Xa and VIIa/TF complex by LACI is shown in FIG. 1.

Coagulation occurs via two pathways: intrinsic and extrinsic. Theintrinsic and extrinsic pathways of coagulation consist of severalproteases that are activated in a series which, unless inhibited, resultin the formation of fibrin clots. LACI acts at two steps in thecoagulation cascade pathway both at the Xa and VIIa/TF level asdescribed above. The activation of tissue factor, which LACI inhibits,is a relatively early event in extrinsic pathway. (LACI has also beencalled Extrinsic Pathway Inhibitor (EPI) and tissue factor pathwayinhibitor (TFPI)). LACI inactivates Factor Xa which is a common proteasefor the extrinsic and intrinsic pathway and is downstream fromactivation of tissue factor.

The concentration of LACI in normal plasma is 100 ng/ml. A report byBajaj et al., 1987, J. Clin. Invest., 79:1874-1878, suggests that LACIis synthesized in liver and endothelial cells and is consumed during DICin patients. Specifically, LACI values in the plasma of 15 healthyvolunteers ranged from 72 to 142 U/ml with a mean of 101 U/ml.Interestingly, LACI levels of 10 patients with DIC were 57±30 U/ml(p<0.001). In contrast, LACI levels of 12 patients with hepatocellulardisease were a mean of 107±33, i.e., similar to normal. Sandset et al.,1989, J. Internal Med., 225:311-316, monitored LACI plasma levels duringa 7-day observation period from patients with pneumonia (n=13), and instroke patients with infarction (n=9), and haemorrhage (n=9). Inpneumonia patients, LACI showed a weak but not significant increase inthe recovery period (p=0.068). In cerebral haemorrhage patients, LACIlevels did not consistently change, while in cerebral infarctionpatients, an increase in LACI levels was observed from day 1 to day 2(p<0.05). This latter effect was most probably due to release of tissuebound LACI by heparin and thus, was only observed in heparin-treatedpatients.

Sandset et al., 1989, Haemostasis, 19:189-195, also serially determinedLACI levels in 13 patients with post-operative/post-traumaticsepticemia. In the survivors (n=8), initial low LACI activity normalizedduring recovery. In the fatal cases (n=5), a progressive increase inLACI activity (maximal 30±15%) was observed until death. The increasemay be explained by a badly damaged endothelium that is releasing thetissue bound LACI into the circulation.

As utilized herein, the term “sepsis” means a toxic condition resultingfrom the spread of bacterial endotoxins from a focus of infection.

As utilized herein, the term “sepsis-associated coagulation disorder”means a disorder resulting from or associated with coagulation systemactivation by a bacterial endotoxin, a product of such bacterialendotoxin or both. An example of such sepsis-associated coagulationdisorder is disseminated intravascular coagulation.

The term “therapeuticaly-effective amount” as utilized herein means anamount necessary to permit observation of activity in a patientsufficient to alleviate one or more symptoms generally associated withsepsis. Such symptoms include, but are not limited to, death, increasedheart rate, increased respiration, decreased fibrinogen levels,decreased blood pressure, decreased white cell count, and decreasedplatelet count. Preferably, a therapeutically-effective amount is anamount necessary to attenuate a decrease in fibrinogen levels in apatient being treated.

LACI Manufacture

LACI can be made and isolated by several methods. For example, cellsthat secrete LACI include aged endothelial cells or young endothelialcells which have been treated with TNF for about 3 to 4 days, alsohepatocytes or hepatoma cells. LACI can be purified from this cellculture by conventional methods. For example, these methods include thechromatographic methods shown in Pedersen et al., 1990, J. of BiologicalChemistry, 265:16786-16793, Novotny et al., 1989, J. of BiologicalChemistry, 264:18832-18837, Novotny et al., 1991, Blood, 78:394-400, Wunet al., 1990, J. of Biological Chemistry, 265;16096-16101, and Broze etal., 1987, PNAS (USA), 84:1886-1890. Furthermore, LACI appears in thebloodstream and could be purified from blood, see Pedersen et al.,supra. However, that method is not suggested or preferred because of thelarge quantities of blood that would be required to obtain sufficientquantities of LACI.

LACI may be produced recombinantly as shown in U.S. Pat. No. 4,966,852.For example, the cDNA for the protein can be incorporated into a plasmidfor expression in prokaryotes or eukaryotes. U.S. Pat. No. 4,847,201,which is hereby incorporated by reference in its entirety, providesdetails for transforming microorganisms with specific DNA sequences andexpressing them. There are many other references known to those ofordinary skill in the art which provide details on expression ofproteins using microorganisms. Many of those are cited in U.S. Pat. No.4,847,201, such as Maniatas, T., et al., 1982, Molecular Cloning, ColdSpring Harbor Press.

The following is an overview about transforming and expressing LACI inmicroorganisms. LACI DNA sequences must be isolated, and connected tothe appropriate control sequences. LACI DNA sequences are shown in U.S.Pat. No. 4,966,852 and it can be incorporated into a plasmid, such aspUNC13 or pBR3822, which are commercially available from companies suchas Boehringer-Mannheim. Once the LACI DNA is inserted into a vector, itcan be cloned into a suitable host. The DNA can be amplified bytechniques such as those shown in U.S. Pat. No. 4,683,202 to Mullis andU.S. Pat. No. 4,683,195 to Mullis et al. (LACI cDNA may be obtained byinducing cells, such as hepatoma cells (such as HepG2 and SKHep) to makeLACI mRNA then identifying and isolating the mRNA and reversetranscribing it to obtain cDNA for LACI.) After the expression vector istransformed into a host such as E. coli the bacteria may be fermentedand the protein expressed. Bacteria are preferred prokaryoticmicroorganisms and E. coli is especially preferred. A preferredmicroorganism useful in the present invention is E. coli K-12, strainMM294 deposited with the ATCC on Feb. 14, 1984, under the provisions ofthe Budapest Treaty. It has accession number 39607. Alternatively, LACImay be introduced into mammalian cells. These mammalian cells mayinclude CHO, COS, C127, Hep G2, SK Hep, baculovirus, and infected insectcells (see also U.S. Pat. No. 4,847,201, referred to above). See alsoPedersen et al., 1990, J. of Biological Chemistry, 265: 16786-16793.

Some specific details about the production of a recombinant proteintypically involves the following:

Suitable Hosts. Control Systems and Methods

First, a DNA encoding the mature protein (used here to include allmuteins); the preprotein; or a fusion of the LACI protein to anadditional sequence which does not destroy its activity or to additionalsequence cleaved under controlled conditions (such as treatment withpeptidase) to give an active protein, is obtained. If the sequence isuninterrupted by introns it is suitable for expression in any host. Ifthere are introns, expression is obtainable in mammalian or othereucaryotic systems capable of processing them. This sequence should bein excisable and recoverable form. The excised or recovered codingsequence is then placed in operable linkage with suitable controlsequences in a replicable expression vector. The vector is used totransform a suitable host and the transformed host cultured underfavorable conditions to effect the production of the recombinant LACI.

Genomic or cDNA fragments are obtained and used directly in appropriatehosts. The constructions for expression vectors operable in a variety ofhosts are made using appropriate replications and control sequences, asset forth below. Suitable restriction sites can, if not normallyavailable, be added to the ends of the coding sequence so as to providean excisable gene to insert into these vectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, or mammalian cells are presently usefulas hosts. Host systems which are capable of proper post-translationalprocessing are preferred. Accordingly, although procaryotic hosts are ingeneral the most efficient and convenient for the production ofrecombinant proteins, eucaryotic cells, and, in particular, mammaliancells are preferred for their processing capacity, for example, theability to form the proper glycosylation patterns. In addition, there ismore assurance that the native signal sequence will be recognized by themammalian host cell, thus making secretion possible, and purificationthereby easier.

Control Sequences and Corresponding Hosts

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species by Bolivar, et al., 1977, Gene, 2:95. pBR322contains genes for ampicillin and tetracycline resistance, and thusprovides additional markers which can be either retained or destroyed inconstructing the desired vector. Commonly used procaryotic controlsequences are defined herein to include promoters for transcriptioninitiation, optionally with an operator, along with ribosome bindingsite sequences, which include such commonly used promoters as thebeta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., 1977, Nature, 198:1056) and the tryptophan (trp)promoter system (Goeddel, et al., 1980, Nucleic Acids Res., 8:4057) andthe λ derived P_(L) promoter and N-gene ribosome binding site(Shimatake, et al., 1981, Nature, 292:128), which has been made usefulas a portable control cassette, as set forth in U.S. Pat. No. 4,711,845,issued Dec. 8, 1987. However, any available promoter system compatiblewith procaryotes can be used.

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used although a number of other strains are commonlyavailable. Examples of plasmid vectors suitable for yeast expression areshown in Broach, J. R., 1983, Meth. Enz., 101:307; Stinchcomb et al.,1979, Nature, 282:39; and Tschempe et al., 1980, Gene, 10:157 andClarke, L., et al., 1983, Meth. Enz., 101:300. Control sequences foryeast vectors include promoters for the synthesis of glycolytic enzymes(Hess, et al., 1968, J. Adv. Enyme Reg., 7:149; Holland, et al., 1978,Biochemistry, 17:4900). Additional promoters known in the art includethe promoter for 3-phosphoglycerate kinase (Hitzeman, et al., 1980, J.Biol. Chem., 255:2073), and those for other glycolytic enzymes, such asglyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other promoters, which havethe additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and enzymes responsible for maltose and galactoseutilization (Holland, supra). It is also believed that terminatorsequences are desirable at the 3′ end of the coding sequences. Suchterminators are found in the 3′ untranslated region following the codingsequences in yeast-derived genes. Many of the vectors illustratedcontain control sequences derived from the enolase gene containingplasmid peno46 (Holland, M. J. et al., 1981, J. Biol. Chem., 256:1385)or the LEU2 gene obtained from YEp13 (Broach, J. et al., 1978, Gene,8:121), however, any vector containing a yeast compatible promoter,origin of. replication and other control sequences is suitable.

It is also, of course, possible to express genes encoding polypeptidesin eucaryotic host cell cultures derived from multicellular organisms.See, for example, Tissue Culture, 1973, Cruz and Patterson, eds.,Academic Press. Useful host cell lines include murine myelomas N51,VERO, HeLa cells, Chinese hamster ovary (CHO) cells, COS, C127, Hep G2,SK Hep, baculovirus, and infected insect cells. Expression vectors forsuch cells ordinarily include promoters and control sequences compatiblewith mammalian cells such as, for example, the commonly used early andlater promoters from Simian Virus 40 (SV40) (Fiers, et al., 1978,Nature, 273:113), or other viral promoters such as those derived frompolyoma, Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses,or immunoglobulin promoters and heat shock promoters. General aspects ofmammalian cell host system transformations have been described by Axel,U.S. Pat. No. 4,399,216, issued Aug. 16, 1983. It now appears also that“enhancer” regions are important in optimizing expression; these are,generally, sequences found upstream of the promoter region. Origins ofreplication may be obtained, if needed, from viral sources. However,integration into the chromosome is a common mechanism for DNAreplication in eucaryotes. Plant cells are also now available as hosts,and control sequences compatible with plant cells such as the nopalinesynthase promoter and polyadenylation signal sequences (Depicker, A., etal., 1982, J. Mol. Appl. Gen., 1:561) are available. Methods and vectorsfor transformation of plant cells have been disclosed in PCT PublicationNo. WO 85/04899, published Nov. 7, 1985.

Host strains useful for cloning and sequencing, and for expression ofconstruction under control of most bacterial promoters include E. colistrain MM294 obtained from E. coil Genetic Stock Center GCSC #6135. Forexpression under control of the P_(L)N_(RBS) promoter, E. coli strainK12 MC1000 lambda lysogen, N₇N₅₃cI857 SusP80, a strain deposited withthe American Type Culture Collection (ATCC 39531), may be used. E. coliDG116, which was deposited with the ATCC (Accession No. 53606) on Apr.7, 1987, may also be used. For M13 phage recombinants, E. coli strainssusceptible to phage infection, such as E. coli K12 strain DG98, can beemployed. The DG98 strain has been deposited with the ATCC (ATCC 39768)on Jul. 13, 1984. Mammalian expression can be accomplished in COS-A2cells, COS-7, CV-1, murine myelomas N51, VERO, HeLa cells, Chinesehamster ovary (CHO) cells, COS, C127, Hep G2, SK Hep, baculovirus, andinfected insect cells. Insect cell-based expression can be in Spodopterafrugiperda.

Transformations

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S. N., 1972, PNAS (USA),69:2110, is used for procaryotes or other cells which containsubstantial cell wall barriers. Infection with Agrobacterium tumefaciens(Shaw, C. H. et al., 1983, Gene, 23:315) is used for certain plantcells. For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, 1987, Virology,52:546 is preferred. Transformations into yeast are carried outaccording to the method of Van Solingen, P. et al., 1977, J. Bact.,130:946 and Hsiao, C. L. et al., 1979, PNAS (USA), 76:3829.

Probing mRNA by Northern Blot: Probe of cDNA or Genoinic Libraries

RNA is fractionated for Northern blot by agarose slab gelelectrophoresis under fully denaturing conditions using formaldehyde,Maniatas, T., et al., 1982, Molecular Cloning, Cold Spring Harbor Press,pp. 202-203, or 10 mM methyl mercury (CH₃HgOH) (Bailey, J. M., et al.,1976, Anal. Biochem., 70: 75-85; Shegal, P. B. et al., 1980, Nature,288:95-97) as the denaturant. For methyl mercury gels, 1.5% gels areprepared by melting agarose in running buffer (100 mM boric acid, 6 mMsodium borate, 10 mM sodium sulfate, 1 mM EDTA, pH 8.2), cooling to 60°C. and adding 1/100 volume of 1 M CH₃HgOH. The RNA is dissolved in 0.5×running buffer and denatured by incubation in 10 mM methyl mercury for10 minutes at room temperature. Glycerol (20%) and bromophenol blue(0.05%) are added for loading the samples. Samples are electrophoresedfor 500-600 volt-hr with recirculation of the buffer. Afterelectrophoresis, the gel is washed for 40 minutes in 10 mM2-mercaptoethanol to detoxify the methyl mercury, and Northern blotsprepared by transferring the RNA from the gel to a membrane filter.

cDNA or genomic libraries are screened using the colony or plaquehybridization procedure. Bacterial colonies, or the plaques for phage,are lifted onto duplicate nitrocellulose filter papers (S&S type BA-85).The plaques or colonies are lysed and DNA is fixed to the filter bysequential treatment for 5 minutes with 500 mM NaOH, 1.5 M NaCl. Thefilters are washed twice for 5 minutes each time with 5× standard salinecitrate (SSC) and are air dried and baked at 80° C. for 2 hours.

The gels for Northern blot or the duplicate filters for cDNA or genomicscreening are prehybridized at 25° to 42° C. for 6 to 8 hours with 10 mlper filter of DNA hybridization buffer without probe (0-50% formamide,5-6×SSC, pH 7.0, 5× Denhardt's solution (polyvinylpyrrolidone, plusFicoll and bovine serum albumin; 1×=0.02% of each), 20-50 mM sodiumphosphate buffer at pH 7.0, 0.2% sodium dodecyl sulfate (SDS), 20 μg/mlpoly U (when probing cDNA), and 50 μg/ml denatured salmon sperm DNA).The samples are then hybridized by incubation at the appropriatetemperature for about 24-36 hours using the hybridization buffercontaining kinased probe (for oligomers). Longer cDNA or genomicfragment probes were labelled by nick translation or by primerextension.

The conditions of both prehybridization and hybridization depend on thestringency desired, and vary, for example, with probe length. Typicalconditions for relatively long (e.g., more than 30-50 nucleotide) probesemploy a temperature of 42° to 55° C. and hybridization buffercontaining about 20%-50% formamide. For the lower stringencies neededfor oligomeric probes of about 15 nucleotides, lower temperatures ofabout 25°-42° C., and lower formamide concentrations (0%-20%) areemployed. For longer probes, the filters may be washed, for example,four times for 30 minutes, each time at 40°-55° C. with 2×SSC, 0.2% SDSand 50 mM sodium phosphate buffer at pH 7, then washed twice with0.2×SSC and 0.2% SDS, air dried, and are autoradiographed at −70° C. for2 to 3 days. Washing conditions are somewhat less harsh for shorterprobes.

Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 μl ofbuffer solution; in the examples herein, typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate. Incubation times of about 1 hour to 2 hours at about 37° C.are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods of Enzymology,65:499-560, 1980.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 minutes at 20° to 25° C. in 50 mM dithiothreitol (DTT)and 5-10 μM dNTPs. The Klenow fragment fills in at 5′ sticky ends butchews back protruding 3′ single strands, even though the four dNTPs arepresent. If desired, selective repair can be performed by supplying onlyone of the, or selected, dNTPs within the limitations dictated by thenature of the sticky ends. After treatment with Klenow, the mixture isextracted with phenol/chloroform and ethanol precipitated. Treatmentunder appropriate conditions with S1 nuclease results in hydrolysis ofany single-stranded portion.

Synthetic oligonucleotides may be prepared by the triester method ofMatteuccei et al., 1981, J. Am. Chem. Soc., 103:3185-3191, or usingautomated synthesis methods. Kinasing of single strands prior toannealing or for labelling is achieved using an excess, e.g.,approximately 10 units of polynucleotide kinase to 1 nmole substrate inthe presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM DTT, 1-2 mM ATP.If kinasing is for labelling of probe, the ATP will contain highspecific activity 32YP.

Ligations are performed in 15-30 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml bovine serum albumin (BSA), 10 mM-50 mM NaCl, and either40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “stickyend” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C. (for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 33-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). Intermolecular blunt end ligations (usuallyemploying a 10-30 fold molar excess of linkers) are performed at 1 μMtotal ends concentration.

In the vector construction employing “vector fragments”, the vectorfragment is commonly treated with bacterial alkaline phosphatase (BAP)in order to remove the 5′ phosphate and prevent religation of thevector. BAP digestions are conducted at pH 8 in approximately 150 mMTris, in the presence of Na²⁺ and Mg²⁺ using about 1 unit of BAP per μgof vector at 60° C. for about 1 hour. In order to recover the nucleicacid fragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors which have been double digested by additional restriction enzymedigestion of the unwanted fragments.

Modification of DNA Sequences

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site specific primer directed mutagenesis isused. This technique is now standard in the art, and is conducted usinga primer synthetic oligonucleotide complementary to a single strandedphage DNA to be mutagenized except for limited mismatching, representingthe desired mutation. Briefly, the synthetic oligonucleotide is used asa primer to direct synthesis of a strand complementary to the phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated in topagar, permitting plaque formation from single cells which harbor thephage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form: 50% will have the original sequence.The plaques are hybridized with kinased synthetic primer at atemperature which permits hybridization of an exact match, but at whichthe mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered.

Verification of Construction

Correct ligations for plasmid construction could be confirmed by firsttransforming E. coli strain MM294, or other suitable host, with theligation mixture. Successful transformants are selected by ampicillin,tetracycline or other antibiotic resistance or using other markersdepending on the mode of plasmid construction, as is understood in theart. Plasmids from the transformants are then prepared according to themethod of Clewell, D. B. et al., 1969, PNAS (USA), 62:1159, optionallyfollowing chloramphenicol amplification (Clewell, D. B., 1972, J.Bacteriol, 110:667). The isolated DNA is analyzed by restriction and/orsequenced by the dideoxy method of Sanger, F., et al., 1977, PNAS (USA),74:5463 as further described by Messing et al., 1981, Nucleic AcidsRes., 9:309, or by the method of Maxam et al., 1980, Methods inEnzymology, 65:499.

Purification of LACI

For purification of mammalian cell expressed LACI, the following methodsmay be used: sequential application of heparin-Sepharose, MonoQ, MonoS,and reverse phase HPLC chromatography. See Pedersen et al., supra,Novotny et al., 1989, J. of Biological Chemistry, 264:18832-18837,Novotny et al., 1991, Blood, 78:394-400, Wun et al., 1990, J. ofBiological Chemistry, 265:16096-16101, and Broze et al., 1987, PNAS(USA), 84:1886-1890. These references describe various methods forpurifying mammalian produced LACI.

Additionally, LACI may be produced in bacteria, such as E. coli, andsubsequently purified. Generally, the procedures shown in U.S. Pat. Nos.4,511,502; 4,620,948; 4,929,700; 4,530,787; 4,569,790; 4,572,798; and4,748,234 can be employed. These patents are hereby incorporated byreference in their entireties. Typically, the heterologous protein (i.e.LACI) is produced in a refractile body within the bacteria. To recoverand purify the protein, the cells are lysed and the refractile bodiesare centrifuged to separate them from the cellular debris (see U.S. Pat.No. 4,748,234 for lowering the ionic strength of the medium to simplifythe purification). Thereafter, the refractile bodies containing the LACIare denatured, at least once (typically in reducing environment), andthe protein is oxidized and refolded in an appropriate buffer solutionfor an appropriate length of time. LACI has a significant number ofcysteine residues and the procedure shown in U.S. Pat. No. 4,929,700should be relevant because CSF-1 also contains a significant number ofcysteine residues. LACI may be purified from the buffer solution byvarious chromatographic methods, such as those mentioned above for themammalian cell derived LACI. Additionally, the methods shown in U.S.Pat. No. 4,929,700 may be employed.

Administration and Formulations

LACI is administered at a concentration that is therapeuticallyeffective to treat and prevent sepsis, acute or chronic inflammation,and other diseases in which cytokines up-regulate tissue factor. Toaccomplish this goal, LACI is preferably administered intravenously.Methods to accomplish this administration are known to those of ordinaryskill in the art.

Before administration to patients, formulants may be added to LACI. Aliquid formulation is preferred. In the example below, LACI wasformulated in 150 mM NaCl and 20 mM NaPO₄ at pH 7.2. However, LACI maybe formulated at different concentrations or using different fornulants.For example, these formulants may include oils, polymers, vitamins,carbohydrates, amino acids, salts, buffers, albumin, surfactants, orbulking agents. Preferably carbohydrates include sugar or sugar alcoholssuch as mono, di, or polysaccharides, or water soluble glucans. Thesaccharides or glucans can include fructose, dextrose, lactose, glucose,mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin,alpha and beta cyclodextrin, soluble starch, hydroxethyl starch andcarboxymethylcelloluose, or mixtures thereof. Sucrose is most preferred.Sugar alcohol is defined as a C₄ to C₈ hydrocarbon having an —OH groupand includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. Mannitol is most preferred. These sugars orsugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to amount used as long as the sugaror sugar alcohol is soluble in the aqueous preparation. Preferably, thesugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %,more preferable between 2.0 and 6.0 w/v %. Preferably amino acidsinclude levorotary (L) forms of carnitine, arginine, and betaine;however, other amino acids may be added. Preferred polymers includepolyvinylpyrrolidone (PVP) with an average molecular weight between2,000 and 3,000, or polyethylene glycol (PEG) with an average molecularweight between 3,000 and 5,000. It is also preferred to use a buffer inthe composition to minimize pH changes in the solution beforelyophilization or after reconstitution. Most any physiological buffermay be used, but citrate, phosphate, succinate, and glutamate buffers ormixtures thereof are preferred. Preferably, the concentration is from0.01 to 0.3 molar. Surfactants that can be added to the formulation areshown in EP Nos. 270,799 and 268,110.

Additionally, LACI can be chemically modified by covalent conjugation toa polymer to increase its circulating half-life, for example. Preferredpolymers, and methods to attach them to peptides, are shown in U.S. Pat.Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546 which are all herebyincorporated by reference in their entirties. Preferred polymers arepolyoxyethylated polyols and polyethylene glycol (PEG). PEG is solublein water at room temperature and has the general formula:R(O—CH₂—CH₂)_(n)O—R where R can be hydrogen, or a protective group suchas an alkyl or alkanol group. Preferably, the protective group hasbetween 1 and 8 carbons, more preferably it is methyl. The symbol n is apositive integer, preferably between 1 and 1,000, more preferablybetween 2 and 500. The PEG has a preferred average molecular weightbetween 1000 and 40,000, more preferably between 2000 and 20,000, mostpreferably between 3,000 and 12,000. Preferably, PEG has at least onehydroxy group, more preferably it is a terminal hydroxy group. It isthis hydroxy group which is preferably activated to react with a freeamino group on the inhibitor. However, it will be understood that thetype and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/IL-2 of the present invention.

Water soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), etc. POG is preferred. Onereason is because the glycerol backbone of polyoxyethylated glycerol isthe same backbone occurring naturally in, for example, animals andhumans in mono-, di-, triglycerides. Therefore, this branching would notnecessarily be seen as a foreign agent in the body. The POG has apreferred molecular weight in the same range as PEG. The structure forPOG is shown in Knauf et al., 1988, J. Bio. Chem. 263:15064-15070, and adiscussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106,both of which are hereby incorporated by reference in their entireties.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in water. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution, and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. A preferred injectable preparationsolution is LACI in an aqueous solution of 150 mM sodium chloride and 20mM sodium phosphate.

While LACI can be administered as the sole active anticoagulationpharmaceutical agent, it can also be used in combination with one ormore antibodies useful for treating sepsis, such as, for example,anti-endotoxin, monoclonal antibodies (endotoxin-binding Mabs) andanti-TNF products such as an anti-TNF murine Mab. LACI can also becombined with interleukin-1 receptor antagonists,bactericidal/permeability increasing (BPI) protein, immunostimulant,compounds having anti-inflammatory activity, such as PAF antagonists andcell adhesion blockers. When administered as a combination, thetherapeutic agents can be formulated as separate compositions which aregiven at the same time or different times, or the therapeutic agents canbe given as a single composition.

LACI may he given in combination with other agents which would beeffective to treat sepsis. For example, the following may beadministered in combination with LACI: antibiotics that can treat theunderlying bacterial infection; monoclonal antibodies that are directedagainst bacterial cell wall components; receptors that can complex withcytokines that are involved in the sepsis pathway; and generally anyagent or protein that can interact with cytokines or complement proteinsin the sepsis pathway to reduce their effects and to attenuate sepsis orseptic shock.

Antibiotics that are useful in the present invention include those inthe general category of: beta-lactam rings (penicillin), amino sugars inglycosidic linkage (aminoglycosides), macrocyclic lactone rings(macrolides), polycyclic derivatives of napthacenecarboxamide(tetracyclines), nitrobenzene derivatives of dichloroacetic acid,peptides (bacitracin, gramicidin, and polymyxin), large rings with aconjugated double bond system (polyenes), sulfa drugs derived fromsulfanilamide (sulfonamides), 5-nitro-2-furanyl groups (nitrofurans),quinolone carboxylic acids (nalidixic acid), and many others. Otherantibiotics and more versions of the above specific antibiotics may befound in Encyclopedia of Chemical Technology, 3rd Edition, Kirk-Othymer(ed.), Vol. 2, pages 782-1036 (1978) and Vol. 3, pages 1-78, Zinsser,MicroBiology, 17th Edition W. Joklik et al. (Eds.) pages 235-277 (1980),or Dorland's Illustrated Medical Dictionary, 27th Edition, W. B.Saunders Company (1988).

Monoclonal antibodies that may be administered along with LACI includethose found in PCT WO 88/03211, to Larrick et al., entitledGram-Negative Bacterial Endotoxin Blocking Monoclonal Antibodies, andU.S. Ser. No. 07/876,854, filed Apr. 30, 1992, to Larrick et al. Bothapplications disclose specific monoclonal antibodies that are useful totreat sepsis and which bind to various antigens on the E. coli bacterialcell wall. A specifically preferred monoclonal antibody is that which isproduced by hybridoma ATCC No. HB9431.

Other agents which may be combined with LACI include monoclonalantibodies directed to cytolines involved in the sepsis pathway, such asthose monoclonal antibodies directed to IL-6 or M-CSF, see U.S. Ser. No.07/451,218, filed Dec. 15, 1989 to Creasey et al. and monoclonalantibodies directed to TNF, see Cerami et al., U.S. Pat. No. 4,603,106;inhibitors of protein that cleave the mature TNF prohormone from thecell in which it was produced, see U.S. Ser. No. 07/395,253, filed Aug.16, 1989, to Kriegler et al.; antagonists of IL-1, such as shown in U.S.Ser. No. 07/517,276, filed May 1, 1990 to Haskill et al.; inhibitors ofIL-6 cytokine expression such as inhibin, as shown in U.S. Ser. No.07/494,624, filed Mar. 16, 1992, to Warren et al.; and receptor basedinhibitors of various cytokine such as IL-1. Antibodies to complement orprotein inhibitors of complement, such as CR₁, DAF, and MCP

After the liquid pharmaceutical composition is prepared, it ispreferably lyophilized to prevent degradation and to preserve sterility.Methods for lyophilizing liquid compositions are known to those ofordinary skill in the art. Just prior to use, the composition may bereconstituted with a sterile diluent (Ringer's solution, distilledwater, or sterile saline, for example) which may include additionalingredients. Upon reconstitution, the composition is preferablyadministered to subjects using those methods that are known to thoseskilled in the art.

As stated above, LACI is useful to therapeutically or prophylacticallytreat human patients with sepsis or septic shock, with or without DIC.Generally, people having sepsis are characterized by high fever (>38.5°C.) or hypothermia (<35.5° C.), low blood pressure, tachypnea (>than 20breaths/minute), tachycardia (>than 100 beats/minute), leukocytosis(>15,000 cells/mm³) and thrombocytopenia (<than 100,000 platelets/mm³)in association with bacteremia. LACI should be administered as soon as apatient is suspected of being septic; presenting themselves with agreater than or equal to 20% drop in fibrinogen or appearance of fibrinsplit products, a rise in the patient's temperature and the diagnosis ofleukopenia, thrombocytopenia and hypotension associated with sepsis.LACI should also be administered when there is a risk of sepsis, forexample, from a gunshot wound, or from a surgical incision. As alsostated above, the preferred route is by intravenous administration.Generally, LACI is given at a dose between 1 μg/kg and 20 mg/kg, morepreferably between 20 μg/kg and 10 mg/kg, most preferably between 1 and7 mg/kg.

Total daily dose administered to a host in single or divided doses maybe in amounts, for example, from about 2 to about 50 mg/kg body weightdaily and more usually 4 to 20 mg/kg, preferably, from about 6 to about10 mg/kg. Dosage unit compositions may contain such amounts orsubmultiples thereof to make up the daily dose. Lower amounts may beuseful for prophylactic or other purposes, for example, from 1 μg/kg to2 mg/kg. The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the patient treated and the particular mode of administration.

The dosage regimen is selected in accordance with a variety of factors,including the type, age, weight, sex, diet and medical condition of thepatient, the severity of the condition, the route of administration,pharmacological considerations such as the activity, efficacy,pharmacokinetic and toxicology profiles, whether a drug delivery systemis utilized and whether the compound is administered as part of a drugcombination. Thus, the dosage regimen actually employed may vary widelyand therefore may deviate from the preferred dosage regimen set forthabove.

Preferably, LACI is given as a bolus dose, to increase circulatinglevels by 10-20 fold for 4-6 hours after the bolus dose. Continuousinfusion may also be used after the bolus dose. If so, LACI may beinfused at a dose between 5 and 20 μg/kg/minute, more preferably between7 and 15 μg/kg/minute.

Generally, LACI may be useful for those diseases that occur due to theup-regulation of tissue factor brought on by TNF, IL-1 or othercytokines. For example, in the examples below, LACI administration isshown to lower the IL-6 concentration. Since IL-6 is one factor that isinvolved in acute or chronic inflammation, LACI administration is usefulfor treating inflammation. Typical inflammatory conditions that can betreated by LACI include: arthritis, septic shock, reperfusion injury,inflammatory bowel disease, acute respiratory disease, trauma, and burn.

In treating chronic or acute inflammation, LACI may be administered inthe same fashion and at the same doses as in the anti-sepsis method.

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

EXAMPLES Example 1

This example illustrates a method for obtaining LACI (TFPI).

Materials

Urea (sequenal grade) and Brij 35 non-ionic surfactant were obtainedfrom Pierce. Mixed bed resin AG501-X8 cation exchanger was purchasedfrom Bio Rad. Mono Q HR 5/5 and HiLoad Q Sepharose anion exchangeresins, and Mono S HR 5/5 and Mono S HR 10/16 cation exchange resinswere obtained from Pharmacia Thromboplastin reagent (Simplastin Excel)was from Organon Teknika Corp. Bovine factor X_(a) and Spectrozyme X_(a)were supplied by American Diagnostica, Inc. SDS-PAGE 10-20% gradient gelwas obtained from integrated Separation Systems.

Methods

Expression Vectors and Cloning Strategies

A full length human TFPI cDNA [Wun et al., J. Biol. Chem. 263, 6001-6004(1988)] was cloned into M13mp18 phage DNA cloning vector as a 1.4 KbEcoRI fragment. Site-directed mutagenesis [Kunkel et al., Proc. Nat.Acad. Sci. USA 32, 488-492 (1985)] was used to introduce an NcoI site atthe initiating ATG. The TFPI gene was then cloned as an NcoI/bluntedMaeIII fragment into pMON5557 with NcoI and blunted HindIII endsresulting in the new vector pMON9308. MaeIII site is 15 bp downstreamfrom the stop codon in the TFPI cDNA. The expression vector containedthe recA promoter, a translational enhancer element and ribosome bindingsite derived from the gene 10 leader of bacteriophage T7 as described byOlins and Rangwala, J. Biol. Chem. 264, 16973-16976 (1989), and the T7transcription terminator. This plasmid also contains an irrelevantsequence, i.e. the bST gene (bovine somatotropin).

The NcoI/NsiI fragment of pMON9308 was then replaced by a synthetic DNAfragment designed to (1) introduce an alanine encoding codon at thesecond position, (2) increase the A-T richness of the 5′ portion of thegene, and (3) improve E. coli codon usage. Four oligonucleotides, twofor each strand, were used. All base substitutions (indicated in uppercase), are silent changes. ECTFPI 2 and 3 were 5′ phosphorylated[Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1982)]. ECTFPI 1 and 2 andECTFPI 3 and 4 were annealed in the kinase buffer by incubating for 5minutes at 70° C. and slow-cooling to room temperature. These fragmentswere cloned into pMON9308 which had been digested with NcoI/NsiI. PCRamplification was used to introduce a HindIII site as well as a TAAtermination codon at the 3′ end of the TFPI gene. The PCR primersTPFIterm and TPFIterm 2 are shown below. The TFPI gene was then moved asa NCOI/HindIII fragment into pMON5766. The resultant plasmid waspMON6870.

N c o           ECTFPI 1 l catggctgattctgaAgaagatgaagaacaTacTa     cgactaagactTcttctacttcttgtAtgAtaatagtgA             ECTFPI 2                         N                          s             ECTFPI3     i                          lttatcacTgatacTgaACtgccaccGctgaaactGatgca     ctatgActTGacggtggCgactttgaCt             ECTFPI 4                     HindIII TFPIterm:   ataaca[aagctt]acatatttt                     NcoI TFPIterm2:  atatat[ccatgg]ctgattctpMON6870 was digested with BglII/HindIII. This fragment, containing theexpression cassette, was cloned into pMON6710 [Obukowicz et al.,Biochemistry 29, 9737-9745 (1990)] which had been digested withBglII/HindIII. The resultant plasmid, pMON6875, includes the tacpromoter, G10 leader from bacteriophage T7, met-ala TFPI, and the p22transcriptional terminator. The plasmids were transformed into MON105(rpoD+rpoH358) containing F′ from JM101 for the expression of TFPIprotein.Fermentation

Ten liter fermentations were run in M9 minimal salts media supplementedwith 20 g/l casamino acids in Biostad E fermentors (B. Braun).Fermentations were run at a temperature of 37° C., 1000 rpm agitation,an air flow rate of 15 l/min and 10 psi backpressure. pH was controlledat 7.0 with ammonium hydroxide. Residual glucose concentration in thefermentation broth was automatically controlled at 1.0+/−0.1 g/l. At anoptical density of 46.0 at 550 nm, the temperature was shifted from 37°C. to 30° C. and isopropyl β-D thiogalactopyranoside (IPTG) was added tothe fermentor to a final concentration of 1.0 mM. The culture washarvested four hours post-induction by concentration in an Amicon DC10Lconcentrator followed by centrifugation in a Beckman J2-21 centrifuge.The 10-liter fermentation yield 335-456 g (average of 376+/−46 g, n=6)wet weight of cell paste. The cell paste was frozen at −80° C. forfurther processing hereinbelow.

Isolation of Inclusion Bodies

Frozen E. coli cell paste was resuspended in cold Milli-Q water at aconcentration of 75 g/l. The cells were thoroughly dispersed with ahomogenizer (Ultra-Turrax model SD-45) for 30 minutes on ice. The cellswere mechanically lysed by three passes through the Manton-Gaulinhomogenizer (model 15M-8TA) at 12,000 psi. Inclusion bodies werecentrifuged in the Sorvall RC-2B centrifuge in the GSA rotor at 10,000rpm (16,270×g) for 20 minutes. The supernatant was discarded. Theinclusion body pellets were collected, resuspended in 1 liter of coldMilli-Q water and dispersed with the Ultra-Turrax homogenizer for 30minutes on ice. The inclusion bodies were cycled through theManton-Gaulin homogenizer two more times on ice. Inclusion bodies werepelleted in the Sorvall RC-2B centrifuge as before. Approximately 60 mgof inclusion bodies were collected for every gram of E. coli cellslysed. The inclusion bodies were stored at −80° C.

Buffer Preparation

All the buffers used for sulfonation and refolding of E. coli TFPIcontained high concentrations of urea. Urea solutions were treated withBio-Rad mixed bed resin AG501-X8 at room temperature for at least 20minutes and filtered through 0.2 μm filter before mixing with buffers.All the solutions used for chromatography were 0.2 μm filtered andsonicated under house vacuum for about 10 minutes.

Sulfonation of Inclusion Bodies

One gm of inclusion bodies (wet weight) was dispersed in 40 ml of asolution containing 50 mM Tris/HCl, pH 8, and 7.5 M urea byhomogenization and vortexing. After the inclusion bodies were largelydissolved, 800 mg of sodium sulfite was added and the mixture was shakenat room temperature for 30 minutes. Then, 400 mg of sodium dithionite or120 mg of sodium tetrathionate was added and the mixture was shaken at4° C. overnight. The solution dialyzed against 800 ml of a solutioncontaining 20 mM Tris/HCl, pH 8, and 6 M urea for more than 5 hours at4° C. using a Spectrapor #2 membrane. The dialyzed solution wascentrifuged at 48,400×g for 1 hour, filtered through a 0.2 μm filter,divided into aliquots, and stored at −80° C.

Anion-Exchange Chromatography of Sulfonated TFPI

On a small scale, the sulfonated and dialyzed inclusion bodies werefractionated on a Mono Q HR5/5 anion exchange column. The column waspre-equilibrated in Q-buffer (20 mM Tris/HCl, pH 8, 6 M urea, 0.01% Brij35 non-ionic surfactant) containing 0.15 M NaCl. Two ml of sulfonatedinclusion bodies were loaded onto the column. The column was washed with15 ml of the equilibration buffer and eluted with a 30-ml gradient (0.15-0.4 M NaCl) in Q-buffer. Fractions of 1 ml were collected. On alarger scale, 40 ml of sulfonated sample (equivalent to 0.56 g of wetweight inclusion body) was loaded onto a HiLoad Q Sepharose 16/10 anionexchange column pre-equilibrated in Q-buffer containing 0.15 M NaCl. Thecolumn was washed with 240 ml of equilibration buffer and then elutedwith a 396-ml gradient (0.15-0.4 M NaCl) in Q-buffer. Nine ml fractionswere collected. Both chromatographies were carried out on a PharmaciaFPLC system at room temperature.

Refold of Sulfonated TFPI

The sulfonated, full-length TFPI pool from anion-exchange chromatographywas diluted to an absorbance of 0.07 O.D. units at 280 nm with Q-buffercontaining 0.3 M NaCl. Solid L-cysteine was added to a finalconcentration of 2 mM. The solution was incubated at room temperaturefor 24 hours, diluted 1:1 with water, 1 mM L-cysteine was added,incubated at room temperature for another 24 hours and then incubated at4° C. for up to 4 to 8 days. pH was maintained at 8.5 by addition of 50mM Tris.

Mono S Chromatography of Refold Mixture

In analytical runs, 2 ml refold mixture was loaded onto a Mono S HR 5/5cation exchange column pre-equilibrated in S-buffer (20 mM sodiumphosphate, pH 6.4, 6 M urea). The column was washed with 10 ml of theequilibration buffer and eluted with a 70-ml gradient consisting of0-0.7 M NaCl in S-buffer. One-ml fractions were collected. Inpreparative runs, the refold mixture was acidified to pH 4.5,concentrated 75-fold, and loaded onto a Mono S HR10/16 anion exchangecolumn pre-equilibrated in S-buffer containing 0.3 M NaCl. The columnwas washed with 15-column volumes of the equilibration buffer and elutedwith a 0.3-0.5 M NaCl gradient in S-buffer.

Tissue Factor-Induced Coagulation Time Assay

Conventional coagulation time assay was performed using a Fibrometer(Becton Dickinson) clot timer. Ninety μl of human pooled plasma wasmixed with 10 μl of TFPI sample or control buffer in the well at 37° C.for 1 min and 0.2 ml of tissue factor (Simplastin Excel, diluted 1:60into a solution containing 75 mM NaCl, 12.5 mM CaCl₂, and 0.5 mg/mlbovine serum albumin) was added to initiate the clotting reaction.

Amidolytic Assay of Factor X_(a) Inhibitory Activity

Inhibitory activity against bovine factor X_(a) of TFPI samples wereassayed by conventional amidolysis of Spectrozme X_(a) as describedpreviously by Wun et al., J. Biol. Chem. 265, 16096-16101 (1990) exceptthat the assay buffer consisted of 0.1 M Tris/HCl, pH 8.4, and 0.1%Triton X-100 non-ionic surfactant.

Protein Determination

The concentration of protein was determined by absorbance at 280 nm andby quantitative amino acid analysis after HCl/vapor phase hydrolysis at110° C. for 24 hours.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Daiichi precasted 10-20% gradient gels were used for SDS-PAGE. Samplesare either unreduced and not boiled or reduced in 3.3% 2-mercaptoethanoland boiled for 3 minutes before electrophoresis. The gels were stainedby Coomassie blue.

Expression of TFPI in E. coli

Three vectors were constructed and used for expression of TFPI in E.coli. The first construct, pMON9308, which contained the original humanTFPI cDNA sequence (except the initiating ATG) and the rec A promoter,achieved a very low level of expression (<0.5% of total cell protein).The second construct, pMON6870, which was similar. to the first but wasaltered by introducing an alanine at the second position, by increasingthe A-T richness of the 5′-end and by improving E. coli codon usage, didnot significantly raise the expression level. The third constructpMON6875, which was similar to the second but used a tac promoter,achieved an expression level of approximately 5-10% of total cellprotein and was used for further tests herein. The majority of TFPI(>90%) appeared to be sequestered in inclusion bodies.

Sulfonation of Inclusion Body and Purification of Full-Length SulfonatedTFPI

In initial tests, it was found that the E. coli lysate or the isolatedinclusion bodies contained very little TFPI activity as measured byanti-factor X_(a) and by tissue factor-induced coagulation time assays.Refolding of TFPI by reduction/re-oxidation and by sulfonation/disulfideinterchange of the crude, solubilized inclusion bodies resulted in verylow recovery of activity. Therefore, attempts were made to purify TFPIprior to refolding step, by sulfonation followed by anion exchangechromatography, taking advantage of the 18 added negatively chargedgroups on the sulfonated TFPI. The sulfonated inclusion bodies werefirst fractionated on an analytical Mono Q HR5/5 anion exchange column.The flow-through and early gradient fractions contained much of thecontaminants E. coli protein and truncated TFPI protein (the latter arelower in molecular weight and are immuno-reactive against anti-TFPI-Ig).The full-length TFPI-S-sulfonate eluted at about 0.28 M NaCl. Thefractionation of sulfonated inclusion bodies was scaled up 20 timesusing a Hiload Q Sepharose 16/10 anion exchanger. The chromatogramappeared somewhat different from that from Mono Q but the fractionationof the full-length TFPI-S-sulfonate appeared comparable as judged fromSDS-PAGE.

Refold of TFPI-S-sulfonate

Sulfonated TFPI underwent spontaneous refolding and oxidation uponmixing with a suitable concentration of L-cysteine. The efficiency ofrefold as reflected in the increase of TFPI activity varies widelydepending on the refold conditions. Numerous refold conditions werecompared and optimized in terms of temperature, pH, urea, L-cysteine andprotein concentration. A 2-stage refold process appeared to be the best.In the first stage, the full-length TFPI-S-sulfonate pool was adjustedto an absorbance at 280 nm of 0.07 O.D. units, 2 mM of fresh L-cysteinewas added, and the mixture was incubated at room temperature for 24hours. During this period, the TFPI activity increased from 0 to about12% of full-length SK hepatoma TFPI which served as a standard forcomparison. In the second stage, the solution was diluted 1:1 withwater, and fresh L-cysteine was added to a final concentration of 1 mM.The mixture was incubated at room temperature for 24 hours, during whichtime the specific activity increased about 2 fold to about 30% that ofSK Hepatoma TFPI. The solution was then left at 4° C. for several daysduring which time the TFPI activity increased.

Fractionation of Refold Mixture by Mono S Chromatography

The specific activity of the refold mixture was lower than the purifiedmammalian SK TFPI which suggests that the former may contain bothcorrectly folded and misfolded molecules or only partially activemisfolded molecules. The refold mixture was fractionated on ananalytical Mono S cation exchange column. When the UV-absorbingfractions were analyzed for TFPI activity, the highest specific activitywas associated with a sharp peak (fraction 52) eluted at 0.52 M NaCl.All the other fractions had a specific activity less than 30% that offraction 52. SDS-PAGE analysis showed that fraction 52 contained a sharpband and all other fractions, together with pre-column refold mixture,consisted of diffuse, multiple bands under nonreducing condition. Thediffuse bands are apparently mainly full-length TFPI in various foldedforms since they become sharp-banded upon reduction (see the last twolanes on the right). By making the gradient more shallow, the resolutionof the peaks become better and all the protein peaks appeared to eluteat lower NaCl concentrations Further, it was possible to wash out themajority of the low-activity peaks with 10 column volumes of 0.3 M NaClbefore eluting the active peak with a shadow gradient.

Based on the above results, the chromatography was scaled up using aMono S HR10/16 cation exchange column. The column was washed with 15column volumes of 0.3 M NaCl which essentially washed out all lowactivity peaks. Afterwards, a shadow gradient eluted a peak of proteinthat contained the active TFPI. SDS-PAGE analysis shows that the peakgave a sharp band under either reducing or non-reducing conditions. Thereduced and boiled protein migrated somewhat slower in SDS-PAGE.

Stoichiometry of the Interaction of Refolded TFPI with Factor X_(a)

Inhibition of bovine factor X_(a) by the active refolded E. coli TFPIwas examined by measuring the residual amidolytic activity usingSpectrozyme X_(a). The molar ratio of TFPI to bovine factor X_(a) thatresulted in the complete inhibition of the latter was 1:1 (open circle).For comparison, the stoichiometry of interaction of SK Hepatoma TFPIwith bovine factor X_(a) was also 1:1 (closed circle).

Inhibition of Tissue Factor-Induced Coagulation

The ability of the active, refolded E. coli TFPI to inhibit tissuefactor-induced coagulation in human plasma was compared with that of thepurified SK Hepatoma TFPI. The activity of the E. coli TFPI wasapproximately two fold more active than SK Hepatoma TFPI on a per molbasis as judged from the concentrations of each TFPI that produce thesame prolongation of clotting time.

TABLE 1 Summary of refold and purification of active E. coli TFPI.Specific Volume Total activity^(a) A₂₈₀ nm (ml) A₂₈₀ nm (Sk unit/mA)Yield Starting — — — — — material 0.56 g inclusion body Sulfonated 6.125 153 0 — inclusion body HiLoad Q pool 0.8 46 37 0 — Refold mixture0.035 1050 37 0.66 100 Mono S pool 0.142 48 6.8 2.0 18 ^(a)Specificactivity was determined by tissue factor-induced coagulation time assayas described in METHODS. One SK unit is defined as the amount ofactivity equivalent to that produced by 1 mA (1 × 10⁻³ absorbance unitat 280 nm or 1.31 ug) of purified full-length SK hepatoma TFPI.

Example 2

This example illustrates the effectiveness of using LACI to treatpatients susceptible to or afflicted with sepsis. In particular, thisexample illustrates the effectiveness of using LACI to treat asepsis-associated coagulation disorder, namely, DIC.

Recombinant TFPI was expressed as a glycosylated protein using mouseC127 cells as host and was purified by chromatography on a monoclonalantibody convalently attached to Sepharose 4B as described by Day et al.[Blood 76, 1538(1990)].

Baboons, 9 month of age weighing 9-13.9 kg, were randomly selected forLACI or excipient pretreatment (1 hr or 15 min) protocol. Each baboon isimmobilized with ketamine hydrochloride, 14 mg/kg intramuscularly on themorning of the study and slowly anesthetized with sodium pentobarbital(˜9 mg/kg) via a percutaneous catheter positioned in the cephalic veinand brachial vein. The femoral artery and one femoral vein arecannulated aseptically to measure aortic pressure, obtain blood samples,and for infusion of LACI, live organisms, isotonic sodium chloride andsodium pentobarbital. Animals were pretreated with LACI [3.5 mg LACI perml of excipient (150 mM sodium chloride and 20 mM sodium sulfate)] orexcipient control as an I.V. bolus 40 μg/kg over 15 minutes and then asan infusion at 5.6 μg/kg/min for 545 minutes in the left cephalic vein.Baboons were challenged at time 0 with either 3 ml/kg (4×10¹⁰) or 4ml/kg (5×10¹⁰) of live E. coli. The actual dosing schedule and groupassignment appear below:

Time of Test Average Article Bacterial # of Animals Administration DoseGroup Males Females (min.) (cfu/kg) 1 1 4 Excipient −60(3) −15(2) 4.1 ×10¹⁰ 2 1 2 LACI −60(3) 3.8 × 10¹⁰ 3 0 2 Excipient −15(2) 5.4 × 10¹⁰ 4 03 LACI −15(3) 4.9 × 10¹⁰ Blood samples were collected at −60 or −15, 0,+60, +120, +240, +360, +600, and +720 minutes for determination oflevels of fibrinogen and fibrin degradation products. Results are shownin Tables 2 and 3.

TABLE 2 Individual Animal Fibrinogen Level (% of Time Zero) +240 +360+720 −60/−15 0 +60 +120 (min.) (min.) (min.) Group 1 100 100 100 67 4023 16 100 100 93 85 16 1 1 100 95 100 68 32 25 15 100 100 100 100 29 8 6100 100 85 64 52 24 20 Average 100.0 99.0 95.6 76.8 33.8 16.2 11.6 STDDEV 0.0 2.0 6.0 13.7 11.9 9.8 7.0 Group 2 100 84 84 64 84 84 84 100 10086 100 86 108 86 100 83 108 108 100 95 108 Average 100.0 89.0 92.7 90.790.0 95.7 92.7 STD DEV 0.0 7.8 10.9 19.1 7.1 9.8 10.9 Group 3 100 100100 100 44 15 8 100 100 100 82 19 7 6 Average 100.0 100.0 100.0 91.031.5 11.0 7.0 STD DEV 0.0 0.0 0.0 9.0 12.5 4.0 1.0 Group 4 100 100 121121 100 100 83 100 90 90 85 79 62 60 100 80 91 80 72 75 64 Average 100.090.0 100.7 95.3 83.7 79.0 69.0 STD DEV 0.0 8.2 14.4 18.3 11.9 15.8 10.0Group 1 = Excipient control (4.1 × 10¹⁰ cfu/kg) Group 2 = LACI (3.8 ×10¹⁰ cfu/kg) Group 3 = Excipient control (5.4 × 10¹⁰ cfu/kg) Group 4 =LACI (4.9 × 10¹⁰ cfu/kg)

TABLE 3 Individual Animal Fibrin Degradation Products (μg/ml) +240 +720−60/−15 (min.) (min.) Group 1 10.00 320.00 320.00 10.00 80.00 320.0010.00 80.00 160.00 10.00 80.00 160.00 10.00 10.00 160.00 Average 10.00114.00 224.00 STD DEV 0.00 106.51 78.38 Group 2 10.00 10.00 10.00 10.0010.00 10.00 10.00 10.00 10.00 Average 10.00 10.00 10.00 STD DEV 0.000.00 0.00 Group 3 10.00 40.00 160.00 10.00 20.00 160.00 Average 10.0030.00 160.00 STD DEV 0.00 10.00 0.00 Group 4 10.00 10.00 20.00 10.0010.00 80.00 10.00 20.00 40.00 Average 10.00 13.33 46.67 STD DEV 0.004.71 24.94 Group 1 = Excipient control (4.1 × 10¹⁰ cfu/kg) Group 2 =LACI (3.8 × 10¹⁰ cfu/kg) Group 3 = Excipient control (5.4 × 10¹⁰ cfu/kg)Group 4 = LACI (4.9 × 10¹⁰ cfu/kg)

There was a clear effect by LACI on fibrinogen levels in the E. colitreated animals. A drop in fibrinogen is prominent in the excipientcontrols (Groups 1 and 3) from 240 minutes (i.e., two hours after theend of bacterial infusion) and on. The drop was substantially preventedby LACI pretreatment when the baboons were challenged with lower dosebacteria (Group 2), and attenuated when the animals are challenged withthe higher dose bacteria (Group 4).

The generation of fibrin degradation products was not detectable inGroup 2, and slowed down and reduced in Group 4 animals as a result ofpretreatment with LACI. The differences in the above coagulationparameters among the groups are not as prominent at 720 minutes possiblydue to the fact that the LACI infusion was stopped at 540 minutes andthat a certain circulating level of LACI may be necessary to maintain aneffect.

In addition to the above analyses, histopathology studies whereintissues of all groups of the above baboons were processed, stained withhematoxylin and eosin, and examined by light microscopy. The kidneys,lungs, adrenals, liver and spleen appeared to be the main organsaffected by the E. coli challenge. Reduced pathology in some targetorgans such as adrenals and kidneys was observed.

Thus, the conclusion drawn from the above is that the effect of LACI onseptic shock is evident, particularly in view of the attenuation of thefibrinogen drop end generation of fibrin degradation products, and thereduced pathology in some target organs, such as the adrenal and kidney.

Example 3

This example illustrates the effectiveness of using LACI to promotesurvival in patients which are susceptible to or afflicted with sepsis.In particular, this example illustrates the effectiveness of using LACIto treat gran-negative sepsis. LACI was prepared by the method describedabove in Example 1.

Male and female Papio anubis baboons (7.6±2.4 kg) from the Charles RiverPrimate Center (Wilmington, Mass.) were quarantined for a minimum ofthirty days in the University of Oklahoma Animal Facility (OklahomaCity, Okla.).

Each baboon was immobilized with ketamine hydrochloride, 14 mg/kgintramuscularly on the morning of the study and slowly anesthetized withsodium pentobarbital (−9 mg/kg) via a percutaneous catheter positionedin the cephalic vein. To compensate for insensible fluid loss, theanimals were infused with isotonic saline at a rate of 3.3 ml/kg/hr for12 hours via the barachial vein in the right leg. LACI or PBS buffercontrol was administered to the animals through the brachial vein 30minutes after the administration of bacteria. LACI was administered at aloading dose over fifteen minutes and simultaneously started acontinuous infusion of LACI for an additional 675 minutes (counting fromstart of bacterial infusion, which was defined as time zero).

E. coli 086: K61H were used to inoculate tryptic soy broth agar (2);viability counts of the inoculum were determined by standard dilutiontechniques. At time zero, baboons received an infusion of 4.5×10¹⁰ livebacteria per kg body weight (4 mls/kg), administered through apercutaneous catheter in the right cephalic vein by continuous infusionfor 2 hours.

The femoral artery and one femoral vein were cannulated aseptically tomeasure mean systemic arterial pressure, obtain blood samples and forantibiotic administration. Gentamicin was given (9 mg/kg i.v.) at end ofE. coli infusion, i.e., at T+120 min. for 30 minutes and then 4.5 mg/kgat T+300 min. and T+540 minutes for 30 min. Gentamicin (4.5 mg/kg IM)was then given at the end of the experiment and once daily for 3 days.

Animals were maintained under anesthesia and monitored continuously for12 hours. Blood samples were collected hourly for hematology, clinicalchemistry, cytokines (TNF, IL-6) and LACI determinations. Similarly,respiration rate, heart rate, mean systemic arterial pressure andtemperature were monitored hourly.

Animals surviving 7 days were considered survivors and subsequentlyeuthanized for necropsy at the 8th day.

See Hinshaw, L. B., Archer, L. T., Beller-Todd, B. K., Coalson, J. J.,Flournoy, D. J., Passey, R., Benjamin, B., White, G. L. Survival ofprimates in LD₁₀₀ septic shock following steriod/antibiotic therapy, J.Surg. Res., 26, 151-170 (1989), and Hinshaw, L. B., Brackett, D. J.,Archer, L. T., Beller, B. K., Wilson, M. F. Detection of the“hyperdynamic state” of sepsis in the baboon during lethal E. coliinfusion, J. Trauma, 23, 361-365, (1982); which are incorporated hereinby reference.

Results of this study are shown in Table 4.

TABLE 4 E. coli LACI Baboon Data Fibrinogen Platelet Ct. Blood PressureBaboon 240′ 720′ 702′ Hemolysis 3 hr 12 hr Recovery at 24 hr. Survival #% of T = O % of T = O 6 hr 12 hr % of T = O Consciousness AlertnessMobility Time Controls  3 <1 <1 27 −− −− 61 41 18 hrs  6 8 <1 21 − − 5184 −− −− −− 33½ hrs 18 4 <1 19 − − 65 84 − − − 66 hrs 19 <1 <1 22 −− −−89 122 − − − 7 days 20 4 <1 26 + + 53 45 − − − 83 hrs Low Dose (LoadingDose 0.7 mg/kg: Maintenance Dose 3.0 ug/kg/min)  7 78 8 37.5 − −− 62 5018 hrs  8 55 50 32.2 − − 38 69 ++ ++ ++ 53 hrs 10 32 10 15 − − 73 63 + +− 7 days 11 93 83 26 + + 63 89 ++ ++ − 7 days 16 43 43 33 − − 75 80 − −− 7 days 17 74 84 30 + + 75 90 ++ + + 7 days High Dose (Loading Dose 1.0mg/kg: Maintenance Dose 9.5 up/kg/min)  4 77 22 70 + + 56 87 + + + 7days  5 79 113 59 + + 75 77 ++ ++ ++ 7 days 12 116 71 48 + + 79 68 ++ ++++ 59.5 hrs 13 86 49 30 + + 61 82 ++ ++ ++ 7 days 14 88 105 30 + + 74 71++ ++ ++ 7 days 15 54 54 41 + + 78 90 ++ ++ ++ 7 days Recovery Code ++Very alert, very mobile + Somewhat alert, slightly mobile − Appearstired, cognizant of surrounding, sitting up, petechia noted −− Lyingdown, not cognizant of surroundings, eyes blinking, petechia notedHemolysis Code − Hemolysis noted + No hemoylsis noted

Example 4

Production of LACI

A. Aged Cells

Human umbilical vein endothelial cells (HuVec) were plated andmaintained in a standard tissue culture medium. They were aged for 32-36days, fed twice a week with fresh medium, and the medium supernatant wasremoved after 32 days (called conditioned medium or CM). The CMcontained LACI.

B. Induced Cells

The same HuVec cells were plated and maintained in a tissue culturemedium for 24-48 hours and then they were contacted with variousconcentrations of tumor necrosis factor (TNF) for 3-4 days. The mediumcontaining LACI was removed and is called TNF CM.

Example 5 LACI Inhibition of Sepsis

The following assay was devised to measure the inhibition of sepsis byLACI. HuVec cells were plated and incubated for 48 hours. Bacteriallipopolysaccharide (LPS) was added as an inducer of sepsis. The additionof LPS was the best way to stimulate a sepsis-like response which wasbroader than simple coagulation. When the inducer was added, a testsample was added to examine its effect on the LPS effect on theendothelial cells. The sample that was tested contained LACI. The cellswere incubated between 4 and 5 hours and then chromozyme was added. Thechromozyme contains Factors II, VII, IX, and X. This first methodmeasured the inhibition of tissue factor induction and inhibition ofactivity. In an alternative of the present assay, which measuresinhibition of tissue factor activity, the sample was added together withthe chromozyme and then incubated for 45 minutes. LACI inhibitoryactivity was measured by reading optical density (due to color changes)in a spectrophotometer at A₄₀₅.

Aged and TNF induced condition medium was prepared as in Example 4. FIG.2 displays a dose dependent inhibition of tissue factor activity by asubstance contained in the respective media shown in the figure. Thenature of the substance was identified by the following experiment whichinvolved inhibition of tissue factor activity determined as follows:HuVec cells were prepared for the assay. One cell sample was leftuntreated as a control. Another cell sample was induced with LPS withoutthe addition of any potential inhibitor. Subsequently, six classes ofsamples were run using aged and TNF condition medium containing LACIwith 0, 10, and 100 mg of LACI antibody. FIG. 3 shows the result of thisexperiment. For example, (Lane 1 starting from the left) was the controland very little tissue factor activity was detected. Lane 2 shows 100%of tissue factor activity and induction by addition of LPS. Lanes 3, 4,and 5 show linearly increasing amounts of activity (and thus induction)depending on the amount of anti-LACI antibody. For example, the 0concentration (Lane 3) showed that very little tissue factor activitywas detectable, suggesting lack of tissue factor induction. Thisindicated that LACI inhibited the activity of the tissue factor inducedby LPS. Lanes 4 and 5 show a similar result, however, the amount oftissue factor activity/induction increased as larger amounts of LACIwere neutralized by the anti-LACI antibody. Lanes 6, 7, and 8 (with TNFconditioned medium) also display a nearly identical magnitude ofinhibition of tissue factor activity as that shown for Lanes 3, 4, and5. To confirm the identity of the substance in the conditioned media, weused various concentrations of highly purified LACI in the absence orpresence of neutralizing antibodies. The results match the findingsutilizing aged and TNF induced conditioned media. See FIG. 4.

These data indicate that LACI will inhibit the effects of LPS on HuVeccells in a concentration dependent manner and this effect may bereversed upon the addition of various concentrations of neutralizingantibodies to LACI. Furthermore, this model proves that LACI can be usedto treat sepsis, and its effects were not simply restricted to itsanticoagulant properties.

Example 6 Treatment of Human Patients Using LACI

Human patients which are affected by sepsis may be therapeuticallytreated by using LACI. When the patient presents themselves withincreased temperature, drop in blood pressure, a decrease in white cellcount, and a drop ≧20% in fibrinogen, LACI is administered intravenouslyas a bolus dose of 3-10 mg/kg and as an infusion of 10-20 μg/kg/min for3-4 hours. Alternatively, LACI may be administered at a continuous rateof approximately 10 mg/kg/min for 3 days or for 4 hours daily for 3-4days. Antimicrobial therapy or broad spectrum antibiotics areadministered to the patient along with the LACI.

LACI is given prophylactically in the same manner.

Example 7

In this experiment, highly purified recombinant LACI (6 mg/kg) wasadministered either thirty minutes or four hours after the start of alethal intravenous E. coli infusion in baboons. Early post treatment ofLACI resulted in a) permanent 7 day survivors (5/5) with significantimprovement in quality of life, while the mean survival time for thecontrols (5/5) was 39.9 hrs. (no survivors); b) significant attenuationsof the coagulation response and various measures of cell injury, withsignificant reductions in pathology observed in E. coli sepsis targetorgans including kidneys, adrenals and lungs. LACI administration didnot affect the drop in mean systemic arterial pressure, the increases inrespiration and heart rate or temperature changes associated with thebacterial infusion. LACI treated E. coil infected baboons had twentyfold lower IL-6 levels than their phosphate buffered saline treatedcontrols. In contrast to the earlier 30 minute treatment, theadministration of LACI at four hours i.e., 240 minutes, after the startof bacterial infusion resulted in prolongation of survival time, withforty percent improvement in survival rate (two survivors) and someattenuation of the coagulopathic response, especially in animals inwhich fibrinogen levels were above 10% of normal at the time of LACIadministration.

Recombinant Tissue Factor Pathway Inhibitor

LACI was expressed in the human hepatoma cell line SK Hep as describedin Wun et al. 1992, Thrombosis Haemost., 68:54-59. Detection ofBacterial Endotoxins with the Limulus Amebocyte Lysate Test, Alan R.Liss, Inc., NY. The material was purified by standard techniques toprovide >95% pure preparations. LACI was formulated in 150 mM NaCl and20 mM NaPO₄ (pH 7.2), which served as the excipient control. Finalprotein concentration in a LACI sample ranged from 2.3-3.7 mg/ml,determined by amino acid composition; endotoxin levels ranged from 8 to27 endotoxin units per 15 milligrams of protein. LACI lots weremonitored for biological activity using a tissue factor inhibition assay(Boze et al., Blood 71:335-343 (1988)).

Baboons

Male and female Papio anubis baboons (7.6±2.4 kg) from the Charles RiverPrimate Center (Wilmington, Mass.) were quarantined for a minimum ofthirty days in the University of Oklahoma Health Sciences Center AnimalResource Facility (Oklahoma City, Okla.). Animals were free ofinfections or parasites with hematocrits ≧36%.

Bacteria

Escherichia coli 086:K61H organisms (ATCC 33985; Rockville, Md.) wereisolated from a stool specimen at Children's Memorial Hospital, OklahomaCity. They were stored in the lyophilized state at 4° C. after growth intryptic soybean agar and reconstituted and characterized as described inHinshaw et al., J. Trauma 23:361-365 (1982).

Assays

Endotoxin Measurement

Endotoxin levels in LACI preparations and the excipient buffer weremonitored by the limulus amebocyte lysate test (Wun et al., Thromb.Haemost. 68:54-59 (1992)). LPS from E. coli (B5505; Mallinckrodt, St.Louis, Mo.) were included as a standard. The detection limit of theassay was 10 endotoxin units (E.U.)/ml.

TNF ELISA

Baboon TNF levels in plasma were measured using an ELISA developed fordetecting human TNF (Creasey et al., Circ. Shock 33:84-91 (1991)): apurified monoclonal anti-TNF antibody (24510E11) was bound to microtiterplate wells (Dynatech Immunolon I, Fisher). Unoccupied binding sites onthe plastic were then blocked with bovine serum albumin (BSA). Aliquotsof standard concentrations of purified recombinant human TNF or baboonplasma samples were incubated in duplicate. ELISA wells were exposed tohorseradish peroxidase (HRP)-conjugated affinity packed polyclonalrabbit antibody to recombinant human TNF followed by 0-phenylenediaminesubstrate as chromogen. Wells were rinsed repeatedly withphosphate-buffered saline solution (PBS, Ph 7.5) between successiveincubations. Optical density (OD) was read on an automateddual-wavelength plate reader at 490 nm (Bio-Tek Instruments). Thedetection limit for baboon TNF in this assay was 0.5 ng/ml.

IL-6 Bioassay

IL-6 bioactivity was quantified in baboon plasma using theIL-6-dependent murine hybridoma cell line B9, using IL-6 commerciallyavailable from Amgen, Inc. (Thousand Oaks, Calif.), as the assaystandard (Creasey et al., supra). The detection limits of this assaywere 10 pg/ml.

LACI Levels

A competitive fluorescent immunoassay for LACI was used as previouslydescribed in Novotny et al., Blood 78:394-400 (1991): a rabbit anti-LACIIgG was used to capture LACI in the sample to be tested and FITC-LACI(HepG2) was added to quantitate the number of anti-LACI binding sitesremaining. Standard curves were constructed using dilutions of pooledhuman plasma (George King Biomedical, Overland Park, Kans.) or of pureHepG2 LACI.

The LACI functional assay (tissue factor-inhibition assay) is athree-stage clotting assay. Briefly, in the first stage, the sample tobe tested is incubated with crude brain tissue factor, factor X, factorVII, and calcium. After 30 minutes of incubation, additional factor X isadded and 1 minute later factor X-deficient plasma is added and time toclot is measured in a fibrometer. Residual factor VII(a)/tissue factoractivity in the second stage of the assay is inversely proportional tothe LACI concentration in the test sample. Thus, prolongation of theclotting time reflects higher LACI activity. Standard curves wereconstructed using dilutions of pure HepG2 LACI.

Pharmacokinetic Analysis

The data for each baboon (μg LACI/ml plasma at various sample times)were fit to a two-compartment model. The model parameters weredetermined by nonlinear least squares curve-fitting procedures using thePKDAAS data analysis system (developed for the VAX computer at ChironCorporation deposited at the U.S. Copyright Office as registration No.TXU 416-977). Corrected concentrations at each time, C(t), were weightedas the reciprocals of each concentration squared. The weighted valueswere then fitted to individual subjects' curves using the followingbiexponential equation:C(t)=(DOSE/VC)*[(1−B)*2^(−t/α) +B*2^(−t/β)],where t is time and VC, B, α, and β are model parameters. The sum of thecoefficients was normalized to 1.0. The systemic clearance (CL) was thencalculated from:CL=VC/MRT, whereMRT=[(1−B)*α+B*β]ln(2).Statistical Analysis

Data were analyzed with the students' t-test to determine significantdifferences (p<0.05) in means between groups at given times. Theanalysis of variance (ANOVA) and the multicomparison Duncan's test wereused to determine significant differences between means at time 0 andsubsequent times within groups. The Fisher's exact test was used todetermine significant differences between groups with respect tosurvival rates.

Pharmacokinetic Studies

To establish the appropriate LACI dosage for the E. coli septic shockmodel, we performed a pharmacokinetic study in three healthy baboons.FIG. 5 shows that administered as a bolus at 0.5 mg/kg, LACI exhibited atwo phase half life; an alpha phase of approximately two minutes and abeta phase of about two hours. These data were then modeled as describedabove to identify the-necessary LACI dosage to achieve a circulatingLACI serum concentration of 2 μg/ml, which was arbitrarily defined asthe desired LACI blood concentration since it has been reported thatendogenous levels of LACI in primates is approximately 0.1 μg/ml(Novotny et al., J. Biol. Chem. 264:18832-18837 (1989)). Thus to achievea 20-fold increase in LACI serum concentrations in the baboons, weadministered LACI at a loading dose of 700 μg/kg and a maintenance doseof 10 μg/kg/min (i.e. a total dose of 6,000 μg/kg) startedsimultaneously, 30 minutes after the start of the E. coli infusion.

Experimental and Infusion Procedures

Each baboon was immobilized with ketamine hydrochloride, 14 mg/kgintramuscularly on the morning of the study and slowly anesthetized withsodium pentobarbital (˜9 mg/kg) via a percutaneous catheter positionedin the cephalic vein as described in Hinshaw et al., J. Surg. Res.28:151-170 (1989). To compensate for insensible fluid loss, animals wereinfused with isotonic saline at 3.3 ml/kg/hr for 12 hours via thebrachial vein 30 minutes or 240 minutes, respectively, after theadministration of bacteria. LACI was administered at a loading dose of700 μg/kg for 15 minutes and a continuous infusion of LACI at 10μg/kg/min was given for an additional 525 minutes (counting from startof bacterial infusion, which was defined as time zero). To deliver thesame total LACI dose per baboon, animals treated at +240 minutesreceived a loading dose of 2.8 μg/kg for fifteen minutes andsimultaneously received a continuous infusion of LACI at 10 μg/kg/minfor 480 min.

E. coil 086:K61H were used to inoculate tryptic soy broth agar, andviability counts of the inoculum were determined by standard dilutiontechniques. At time zero, baboons received an infusion of ≧4.5×10¹⁰ livebacteria per kg body weight (4 ml/kg), administered through apercutaneous catheter in the right cephalic vein by continuous infusionfor 2 hours.

The femoral artery and one femoral vein were cannulated aseptically tomeasure mean systemic arterial pressure, obtain blood samples and forantibiotic administration. Gentamicin was given (9 mg/kg i.v.) at theend of E. coli infusion, i.e., at T+120 for 30 minutes and then 4.5mg/kg at T+360 and T+540 minutes for 30 min. Gentamicin (4.5 mg/kg IM)was then given at the end of the experiment and once daily for 3 days.

Animals were maintained under anesthesia and monitored continuously for12 hours. Blood samples were collected hourly for hematology, clinicalchemistry, cytokines (TNF, IL-6, and LACI determinations. Similarly,respiration rate, heart rate, mean systemic arterial pressure andtemperature were monitored hourly. Animals were continuously observedfor the first 30 hours of the experiment. Those surviving 7 days wereconsidered permanent survivors and were subsequently euthanized withsodium pentobarbital for necropsy at the 8th day.

Ten baboons (5 LACI treated and 5 excipient controls) were intravenouslyadministered 2 hour lethal infusions of E. coli. Table 5 shows that LACIrescued five of five E. coli treated baboons who became permanentsurvivors. The mean E. coli dosage of the LACI treated was 5.7×10¹⁰CFU/kg and all animals survived more than 7 days. The mean E. colidosage of the excipient control group was 5.5×10¹⁰ CFU/kg and the meansurvival was 39.9 hours (Table 5). The mean weight of the excipientcontrol group was 8.4 kg (range 5.9 to 12.1 kg) and that of the LACItreated was 6.8 kg (range 5.2 to 8.0 kg). Two females and three malescomposed the excipient control group, while the LACI treated groupconsisted of five males. There was no difference in the mean dose of E.coli administered to each group (p>0.05) nor in the animals' weights(p>0.05).

LACI treated baboons moved about the cage energetically, consumed somefood and drank water normally within 24 hours of receiving lethal E.coli (LD₁₀₀). The excipient control baboons, however, were verylethargic, appeared to have difficulty breathing and exhibited multiplepetechiae over their bodies indicating the occurrence of DIC in thedermal microvasculature.

Coagulation and Hematological Responses to LACI Administration at +30Minutes

To determine the mechanism by which LACI protected the bacteriallyinfected baboons we measured selected physiologic parameters associatedwith coagulation, clinical chemistries and the inflammatory response.FIG. 6 shows that many of the coagulopathies associated with thebacterial infection were inhibited and/or attenuated in the LACI treatedbaboons. Fibrinogen levels in excipient control animals dropped byapproximately 80% by 3 hours, while the LACI treated baboons experiencedonly a 20% drop (p<0.0001). Similarly, the rise in fibrin degradationproducts at 240 and 720 minutes, as a marker of fibrinogen consumption,was not evident in the LACI treated animals as compared to the controls(p<0.05).

Activated partial thromboplastin time (APTT) and prothrombin time (PT)were extremely prolonged at times beyond four hours in the excipientcontrols (FIG. 6). APTT increased from 37 to 208 and then to 226 secondswhile PIT increased from 14 to 58 and then to 137 seconds, at four and12 hours, respectively. In contrast, APT increased from 32 to 45 to 60seconds at four and 12 hours, respectively, and PT increased from 15 to18 seconds to 22 seconds at four and 12 hours, respectively, in the LACItreated baboons (p<0.05).

A gradual drop in platelet cell concentration was noted in the excipientcontrols and in the LACI treated baboons over the 12 hour observationperiod (FIG. 6). LACI treatment, however, retards the drop and is mostapparent at ≧4 hours. The mean platelet concentration of the controlgroup at four, six and twelve hours were 102.8±26, 69±20 and 43±5.0. Incontrast, the mean platelet concentration of the LACI treated group atthe same times were 249±44, 236±35, and 153±31, respectively.

Despite the lack of visible hemolysis in the LACI treated plasmasamples, the hematocrit decreased with time and was lower at 12 hours inthe experimental (treated) group, 36±2%, as compared to the controlgroup, 44±2.% (p<0.05). Furthermore, the mean 7 day hematocrit value ofthe survivors was also low as compared to baseline: 28±1% versus 42±0%.

Consistent with the hematocrit results the red blood cell concentrationdropped only slightly over the initial 12 hours in both the control(4.94±21 to 4.4±0.11) and LACI treated groups (5.20±0.10 to 4.88±0.17),and a low (3.42±0.2×10⁶) red cell concentration was observed in thesurvivors.

Leukopenia occurred to the same degree in the LACI treated and controlgroup, the lowest values (˜1.48×10³/μl) recorded at 2 hours; however,the white blood cell concentration was found elevated at 7 days in thesurvivors with a mean of 19.2±3.5 as compared to the base line of9.0±1.5×10³/μl.

Clinical Responses to LACI Administration at +30 Minutes

Respiration and heart rate increased in both groups. Respiration raterose quickly after the start of the bacterial infusion and remainedelevated for the 12 hour period. Similarly, heart rate increaseddramatically, from 120 beats/min to 200 beats/min, within the first twohours of E. coli infusion and remained elevated during the 12 hours.

Mean systolic arterial pressure (MSAP) and temperature equally declinedin the LACI treated and control groups. A dramatic decrease in MSAP wasobserved at the end of the bacterial infusion. MSAP declined from 107±5mm Hg to 69±5 at two hours and then gradually returned to 93±11 by 10 to12 hours in the control group. Similarly, MSAP declined from 115±9 to74±3 at 2 hours and rose to 85±7 from 6 hours on to 12 hours. The tenbaboons had a decreased temperature response to the E. coli infusion.The mean excipient control temperature at the start of the experimentwas 37.3±0.1° C. and declined slowly to 34.7±2.2° C. at 12 hours. Themean LACI treated temperature was initially 37.0±0.3° C. and changedminimally over the 12 hours where it was 36.9±0.2° C.

Blood Chemistries

Table 6 summarizes clinical chemistries of the E. coli infected andtreated ten baboons. Increases in serum creatinine, total bilirubin,uric acid, lactic acid, triglycerides, anion gap, chloride and sodiumwere measured at 12 hours. The magnitude of the increases, however, waslower in the LACI treated animals than the excipient controls (p<0.05).Changes in the concentrations of the following parameters were observed:albumin, alkaline phosphatase, AST, BUN, calcium, cholesterol, CK,carbon dioxide, cortisol, potassium, lactic dehydrogenase, phosphorous,SGPT and total protein. Their increases or decreases in concentrationwere not affected by the LACI treatment (p>0.05). However, the meanconcentrations of albumin, urea nitrogen (BUN) and lactate did notreturn to baseline values in the LACI treated animals (i.e. thesurvivors) at 7 days. Specifically, albumin concentrations were 2.7±0.2at 7 days as compared to 3.7±0.1 at the start of the experiments. Thusalbumin was reduced by about 25%. Similarly, serum values of ureanitrogen (BUN) at 7 days was 13.8±2.1 versus 29.6±3.9 at the beginningof the experiment. Finally, lactate concentrations were increased byabout 3-fold in the survivors. The mean baseline lactate concentrationsof these animals was 1.7±0.5 meq/L at the start of the procedure andincreased to 5.7±1.2 meq/L at 7 days.

Increases in glucose concentration were observed within two hours inboth groups (p<0.05). Mean values fell gradually beyond the initialincrease but remained consistently higher in the LACI treated animals(p<0.05) until 12 hours. Increases in arterial pH occurred in bothgroups.

TNF and IL-6 Levels

Plasma TNF concentrations were elevated in both the excipient group andLACI treated baboons. Consistent with our previous studies (Creasey etal., Circ. Shock (1991) 33:84-91), peak TNF levels were at 120 min, i.e.at the end of E. coli infusion. LACI treatment did not appear to affectthe rise in serum TNF concentrations nor the kinetics of its release(Table 7). Plasma IL-6 concentrations also increased with time in theexcipient control group. where IL-6 levels started at 26-39 picogramsand rose to 100-200 nanograms beyond four hours (Table 8).Interestingly, plasma IL-6 concentrations in the LACI treated animalswere lower than those of the control group, especially at and beyondfour hours. IL-6 concentrations were about 20-fold lower in the LACItreated than the excipient controls at 12 hours (p<0.05).

Administration of LACI at +240 Minutes

To determine the time beyond which LACI may no longer be effective inattenuating the E. coli shock, we delayed the administration of LACI totwo hours after the end of the bacterial infusion. Fibrinogenconsumption and the generation of fibrin degradation products were to beclearly evident at four hours. Table 8 shows that the mean E. colidosage of the excipient control group in this series of experiments was5.68 (±2.6)×10¹⁰ CFU/kg and the mean survival time of 28.2±9.6 hours.The mean E. coli dosage of the LACI group was 5.43 (±0.19)×10¹⁰ CFU/kgand the mean survival time of 99±29 hours. Two of the five LACI treatedanimals were 7 day survivors (p<0.05). There was no difference in themean weight or E. coli dosage administered to each of the above groups(p>0.05).

Biological and Biochemical Effects of Administration of LACI at +240Minutes

The administration of LACI two hours after the end of the two hourbacterial infusion was effective in slightly attenuating thecoagulopathic response as evident by decreases in FDP levels, andprothrombin time at ≧12 hours. Consistent with +30 minutes, IL-6 levelswere two-fold lower in the LACI treated baboons than their excipientcounterparts at 12 hours. No significant differences in fibrinogenconcentrations, APTT and platelet cell concentration were noted at 12hours between the excipient control and the LACI treated baboons.However, fibrinogen levels at day 7 in the two animals that survivedwere slightly elevated; FDP, APTT and PT values were back close tonormal while platelet cell concentrations were normal in one (435) andlower in the other (97).

Although the red blood cell count and hematocrit fluctuated slightlyover time in both groups during the first 12 hours, the two survivorshad lower hematocrits at day 7 (35 and 19%) as compared to the start ofthe procedure (43 and 41%). Similarly, red blood cell concentration was4.0 and 2.7×10⁶/mm³ on day 7 versus 4.7 and 4.5×10⁶/mm³ at day 0.

Clinical chemistries were measured for the ten baboons comprising theplus four hour study as we had performed for the plus thirty minutestudy. We observed minimal differences between the excipient control andLACI treated baboons at twelve hours. However, consistent with the plus30 minute study, lactate levels were higher in the LACI treated than thecontrols (p<0.05) at 12 hours and remained elevated in the two thatsurvived 7 days (13.2 and 4.0 mg/dl versus 0.5 and 0.6 mg/dl at time 0).In contrast, uric acid levels were slightly lower in the LACI treatedgroup than the controls at 12 hours and returned to normal levels in thetwo LACI treated survivors.

Similar to the plus 30 minute study, all the animals treated at 240minutes experienced leukopenia, and a gradual but small rise in WBCcount over the twelve hours. Furthermore, the two 7 day surviving LACItreated baboons had elevated WBC counts (12.5 and 21.8×10³ cells/mm³) atday 7 as compared to 5.1 and 8.0×10³ cells/mm³ at time zero; this trendis similar to that observed in the survivors of the baboons treated at+30 minutes with LACI.

Pathological Results

Post-mortem examinations were conducted on all baboons. Surveillance ofanimals was continuous for the first 36 hours; consequently tissues wereremoved for analysis within minutes after death thereby avoidingpost-mortem autolytic changes. Lungs, liver, adrenals, kidneys, spleen,and gall bladder were target organs of the E. coli bacterial infusion.Specifically, animals that received excipient +E. coli suffered fromsevere congestion, hemorrhage, fibrin deposition, edema and massiveaccumulation of leukocytes in the lungs and liver, severe congestion ofmedullary sinusoids in the spleen and significant evidence of tubularnecrosis and thrombosis within the kidneys and severe corticalcongestion in the adrenals. Organs not affected by E. coli were stomach,heart, pancreas and small and large intestines. LACI protected theliver, adrenals, kidneys, spleen and gall bladder in which only mild tono pathology were observed. The degree of protection was slightlydiminished in the lungs, in which moderate vascular congestion, and mildleukocyte accumulation were observed.

Results from the present study demonstrated that LACI rescued onehundred percent of the baboons given LD₁₀₀ doses of E. coil whenadministered thirty minutes after the start of the bacterial infusionwhen more than 1×10¹⁰ organisms/kg had already been introduced into theblood of the baboons. In addition, LACI rescued forty percent of thebaboons when given two hours after the end of the bacterial infusioni.e. when greater than 5×10¹⁰ organisms/kg had been infused and many ofthe baboons' host defense mechanisms had been triggered for two hours.

TNF levels peaked at the end of the E. coli infusion i.e. at two hours,while IL-1β and IL-6 levels started to appear (Creasey et al., Circ.Shock 33:84-91 (1991)); the decline and consumption of fibrinogen andgeneration of fibrin degradation products become more easily detectablebetween three and four hours (De Boer, J. P. et al., Circ. Shock (Inpress 1992)). This study shows that LACI could prevent, slow down andeven reverse the consumption of fibrinogen, when administered as late asfour hours after the start of a lethal bacterial infusion.

In addition to attenuating coagulation, LACI attenuated the degree ofcell injury (creatinine, uric acid, lactic acid) and metabolic acidosis(anion gap, chloride and sodium) so clearly evident in the controls.Consistent with the decreased serum levels of many of these markers ofhypoxia, acidosis and cell injury, LACI afforded remarkablemorphological protection to kidneys, adrenals, liver, spleen and thelungs from pathological changes. The efficacy of LACI in baboonschallenged with lethal E. coli shows gram-negative shock is an acuteinflammatory disease of the vascular endothelium and that significantbenefit is achieved by transiently protecting the endothelium frominsults associated with gram-negative bacteria.

Previous studies have shown that within the first 30 minutes of thebacterial infusion, the PMN leukocyte concentration in circulating bloodfell sharply Taylor et al., Colloquium Mosbach Molecular Aspects ofInflammation (1991) Springer Verlag, Berlin Heidelberg, pp. 277-288),thrombin-antithrombin (TAT) complexes, tissue plasminogenactivator/plasminogen activator inhibitor (t-PA/PAI) and plasmin antiplasmin (PAP) complexes had started to appear (De Boer, J. P. et al.,Circ. Shock (1992) In press), and the activation of the complementcascade in lethal E. coli challenge was clearly evident (De Boer, J. P.et al., submitted). LACI treatment resulted in the prevention of tubularnecrosis and glomerular thrombosis in the kidneys; cortical congestion,hemorrhage, necrosis and leukocyte accumulation in the adrenals;prevention of vascular congestion and accumulation of leukocytes in theliver; prevention of medullary congestion, hemorrhage and necrosis inthe spleen; and fibrin thrombi deposition and edema formation in thelungs. LACI significantly attenuated leukocyte influx and vascularcongestion in the lungs. The two baboons that received LACI at fourhours and survived seven days showed a very similar prevention ofpathological changes as those described above. However, there was somemild edema and fibrin present in alveolar sacs of the lungs withmoderate leukocyte accumulation and vascular congestion. There was noevidence of multiple organ failure in any of the LACI treated baboonsthat survived seven days. This degree of protection is remarkable andunexpected given the delayed administration of LACI and the massivebacterial challenge afforded to the baboons.

The LACI-treated, E. coli challenged, 7 day survivors demonstrated alower red blood cell concentration and an increase in leukocyteconcentration. Histological examination did not reveal the occurrence ofhemorrhage in any tissue. Thus the lower hematocrit may be attributedeither to hemodilution or to the slow generation of erythrocytes in thebone marrow. LACI toxicology studies with uninfected baboons may benecessary to resolve this matter.

The decreased IL-6 levels observed in the E. coli challenged and LACItreated baboons in the present study show was unexpected and suggestthat LACI either directly or indirectly exhibits an effect-on theinflammatory response. Thus, in addition to its anticoagulant activity,a physiologic role of LACI is useful in the modulation of theinteraction of the coagulation pathway with various participants of theimmune system.

TABLE 5 Weight, Sex, E. coli Dose and Survival Times of Control andLACI* Treated Baboons at +30 min** Weight Mean Dose E. coli Survival(kg) Sex (CFU/kg × 10¹⁰) Time (hrs) Control (E. coli + excipientcontrol) 26 12.1 M 5.71 46 27 9.8 F 5.60 52.5 32 6.4 F 5.23 9.7 37 7.7 M5.26 30.5 41 5.9 M 5.70 60.5 Mean (± SE) 8.4 ± 1.1 5.50 ± 0.11 39.9 ±9.0 Experimental (E. coli + LACI) 29 8.0 M 4.84 >168 30 7.5 M 5.22 >16831 7.3 M 6.05 >168 38 5.2 M 6.21 >168 40 6.1 M 6.15 >168 Mean (± SE) 6.8± 0.5 5.69 ± 0.28  168 ± 0.0 *LACI - Tissue Factor Pathway Inhibitor**LACI administered 30 min after the onset of a 2 hr infusion of E. coli

TABLE 6 Clinical Chemistry Summary of LACI Treated and Control Baboonsat +30 min** Control (Mean ± STD error) LACI [Mean ± STD error) T 0 T +12 hrs T 0 T + 12 hrs +7 days p < 0.05: Creat (mg/dL) 0.64 ± 0.05 2.68 ±0.27 0.64 ± 0.09 0.92 ± 0.07 0.48 ± .07  T Bili (mg/dL) 0.16 ± 0.02 1.35± 0.33 0.14 ± .02  0.30 ± 0.11 0.20 ± 0.05 Uric Acid (mg/dL) 0.38 ± 0.070.93 ± 0.18 0.50 ± 0.0  0.50 ± 0.00 0.32 ± 0.07 Lactate (mEq/L) 0.94 ±0.38 6.05 ± 0.59 1.74 ± 0.47 4.10 ± 0.34 5.70 ± 1.21 Triglycerides 64 ±7  283 ± 19  101 ± 25  161 ± 28  130 ± 42  (mg/dL) Anion GAP (mEq/L)13.4 ± 0.75 19.25 ± 0.63  11.2 ± 1.24 11.25 ± .25  12.75 ± 1.03  Cl(mEq/L) 107.68 ± 3.73  109.58 ± 1.16  105.76 ± 0.52  117.62 ± 1.08 100.56 ± 6.61  Na (mEq/L) 150.58 ± 4.81  149.50 ± 0.65  146.04 ± 1.31 153.0 ± 1.0  142.26 ± 8.51  p > 0.05 Alb (g/dL) 3.82 ± 0.27 2.90 ± 0.333.66 ± 0.13  2.8 ± 0.06 2.68 ± 0.22 Alk phosp. (IU/L) 827 ± 59  949 ±62  933 ± 68  1032 ± 121  937 ± 117 AST (U/L) 40 ± 3  1531 ± 783  45 ±5  710 ± 484 68 ± 8  BUN (mg/dL) 19.4 ± 1.9  39.0 ± 4.1  29.6 ± 3.9 34.0 ± 3.2  13.8 ± 2.1  CA (mg/dL) 9.9 ± 0.4 7.1 ± 0.3 10.3 ± 0.2  7.9 ±0.2 9.1 ± 0.5 Chol (mg/dL) 126 ± 10  94 ± 7  130 ± 3  86 ±3    135 ± 16 CK (U/L) 604 ± 130 5979 ± 1705 795 ± 348 5594 ± 732  289 ± 79  CO₂(mEq/L) 29.6 ± 2.0  20.4 ± 1.7  29.2 ± 0.9  23.7 ± 0.6  28.2 ± 1.9 Cortisol (μg/dL) 48.4 ± 7.9  120.2 ± 21.1  48.2 ± 12.2 110.5 ± 16.2 28.5 ± 2.6  K (mEq/L) 3.90 ± 0.14 4.75 ± 0.44 3.86 ± 0.14 4.32 ± 0.103.62 ± 0.25 LDH (IU/L) 311 ± 46  3956 ± 1112 317 ± 40  1819 ± 621  433 ±58  Phos (mg/dL) 5.94 ± 0.69 8.28 ± 0.59 4.78 ± 0.34 7.66 ± 0.67 3.96 ±0.55 SGPT (IU/L) 69 ± 23 936 ± 507 49 ± 9  366 ± 281 101 ± 11  TotalProtein (g/dL) 7.00 ± 0.36 5.88 ± 0.35 6.62 ± 0.21 5.36 ± 0.18 5.92 ±0.36 *LACI = Tissue Factor Pathway Inhibitor **LACI administered 30 minafter the onset of a 2 hr infusion of E. coli

TABLE 7 Individual Animal IL-6 Levels (ng/ml) LACI Administration at +30min T 0 +30 +120 +240 +360 +720 Control (E. coli + excipient control) 26.034 .027 21.5 102.3 347.2 468.5 27 .018 .047 27.6 58.4 88.7 31.1 32.010 .020 35.6 217.6 321.7 NT 37 .038 .048 36.7 97.6 196.9 183.2 41 .028.052 32.3 101.7 100.4 63.4 Mean ± SE .03 ± .01 .04 ± .01  30.7 ± 2.8 116± 26.8 211 ± 57.5 187 ± 63.4 Experimental (E. coli + LACI) 29 NT NT 30.057.3 50.8 12.5 30 .150 .639 64.2 51.1 26.1 7.1 31 .013 .030 31.8 48.036.7 10.7 38 .034 .049 16.5 42.7 30.6 6.4 40 .059 .058 17.3 24.8 23.711.3 Mean ± SE .06 ± .03 .19 ± .129 32.0 ± 8.7 44.8 ± 5.5   33.6 ± 4.8  9.6 ± 1.2  NT = Not Tested

TABLE 8 Weight, Sex, E. coli Dose and Survival Times of Control andLACI* Treated Baboons at +240 min** Weight Mean Dose E. coli Survival(kg) Sex (CFU/kg × 10¹⁰) Time (hrs) Control (E. coli + excipientcontrol) 33 6.8 M 6.22 63.5 45 7.7 F 6.29 18.0 46 9.1 M 4.94 32.5 47 6.6M 5.26 9.0 48 7.1 M 5.70 18.0 Mean (± SE) 7.5 ± 0.5 5.68 ± .26  28.2 ±9.6  Experimental (E. coli + LACI) 34 5.2 M 5.65 58 35 7.3 M 5.62 >16836 6.8 M 4.84 >168 44 9.1 M 5.87 69 49 7.5 F 5.17 35 Mean (± SE) 7.2 ±0.6 5.43 ± 0.19 99.6 ± 28.5 *LACI = Tissue Factor Pathway Inhibitor**240 min after the onset of a 2 hr infusion of E. coli

The present invention has been described with reference to specificembodiments. However, this application is intended to cover thosechanges and substitutions which may be made by those skilled in the artwithout departing from the spirit and the scope of the appended claims.

1. A method for treating inflammation comprising administering to apatient a therapeutically effective amount of a protein selected fromthe group consisting of mature TFPI (SEQ ID NO: 5), ala-TFPI,ala-TFPI-1-160, ala-TFPI-13-161, and ala-TFPI-22-150, thereby treatingthe inflammation.
 2. A method in accordance with claim 1, wherein theprotein is chemically conjugated to a polymer consisting essentially ofPEG or POG.
 3. A method in accordance with claim 1, wherein the proteinis administered at a dose between 1 μg/kg to 20 mg/kg.
 4. A method inaccordance with claim 1, wherein the protein is administered at a dosebetween 20 μg/kg to 10 mg/kg.
 5. A method in accordance with claim 1,wherein the protein is administered at a dose between 1 to 7 mg/kg.
 6. Amethod in accordance with claim 1 wherein the inflammation is chronicinflammation.
 7. The method of claim 1 wherein the protein is ala-TFPI.